The desalination plant would be located in the Atacama Region (Chile), where the high solar radiation level justifies an off-grid installation using photovoltaic panels. We classified the collected information about conventional and emerging technologies for seawater desalination, and then an expert panel evaluated these technologies considering five categories: (1) technical characteristics, (2) scale-up potential, (3) temperature effect, (4) electrical supply options, and (5) economic viability. Further, the potential inclusion of graphene oxide and aquaporin-based biomimetic membranes in the desalinization processes was analyzed. The comparative analysis lets us conclude that nanomembranes represent a technically and economically competitive alternative versus RO membranes. Therefore, a profitable desalination process should consider nanomembranes, use of an energy recovery system, and mixed energy supply . This document presents an up-to-date overview of the impact of emerging technologies on desalinated quality water, process costs, productivity, renewable energy use, and separation efficiency.The aim of this work was to study different desalination technologies as alternatives to conventional reverse osmosis (RO) through a systematic literature review. An expert panel evaluated thermal and membrane processes considering their possible implementation at a pilot plant scale (100 m Desalination is a separation process intended to increase water availability in structurally water-deficient countries that suffer recurrent periods of drought. Recently, the International Desalination Association (IDA)  reported3/d, representing 9.81% of the worldwide capacity, followed by the United Arab Emirates, the United States of America, Spain, and China, at 7.5, 4.7, 3.6, and 3.0%, respectively. Installation of desalination plants is mostly preferred when there is no simple alternative to obtain fresh water, low-cost energy is feasible, and high standards of living allow it [Saudi Arabia has the largest water-production installed capacity, with 12 MmNowadays, the greatest challenge for desalination processes is to lower operating and energy costs, through emerging technologies ,11. ThesIn recent years, the number of publications on technoeconomic studies about desalinization processes has increased considerably. For instance, Arafat  related 3/d of purified water) starting from seawater at 20 °C and average salinity of 34,000 ppm.This article presents different desalination technologies as alternatives to conventional reverse osmosis through current state-of-the-art desalination processes considering the existing conventional and emerging technologies and a technical economic comparison between them. Further, this document offers evaluated thermal and membrane processes considering their possible implementation at a pilot plant scale  and the physicochemical process involved . Said grThe main difference between the MED and MSF processes is that while vapor is created in an MSF system through flashing, evaporation of feed water in MED is achieved through heat transfer from the steam in condenser tubes into the source water sprayed onto these tubes. This heat transfer at the same time results in vapor condensation to freshwater . MVC and3) [3/d [3/d [RO is the most energy-efficient technology for desalination, with much lower energy consumption than other technologies (SEC < 3.1 kWh/m3) ,44,45. T3) [3/d ,46. Exte3) [3/d . On the 3/d [3/d ,49.3/d [Electrodialysis (ED) is a membrane-based desalination technology that operates due to an electric gradient, used for many years on brackish water . Typical installed capacities range between 5000 and 425,000 m3/d ,52.The main innovation in emerging desalination technologies relates to the use of renewable energy sources coupled to desalination processes. The most popular renewable energies included in these processes are solar and geothermal energy. For example, Prajapati et al.  investigSD operates with direct or indirect solar energy . Direct It follows that the indirect systems are more efficient and therefore are more adequate for use in industrial scale production.Geothermal energy extraction has the advantage of being independent of the season and climate changes. Geothermal energy sources (GESs) can be applied in both membrane and thermal desalination processes, depending on the location, and on the physical and chemical characteristics of geothermal water. GD recovers heat from a GES to evaporate seawater.Membrane processes integrated with GES are still under development see . Gude 7 investigThe most relevant innovations refer to the synthesis or modification of polymeric materials that allow increasing the productivity and selectivity of desalination processes. Similarly, innovations in conventional technologies were developed, combining mass transport mechanisms, separation gradients, and renewable energy sources. In all cases, the objective is to obtain a reliable desalinated water production technology at the lowest cost.NMs are membranes that contain nanoparticles  in the active layer of the polymer matrix, e.g., polymerized polyamide, aiming at improving hydrophilicity, productivity, and salt rejection .2 due to high chemical stability, low toxicity, defouling, and photocatalytic properties and availability [NMs, also known as thin film nanocomposite membranes, are prepared by the phase inversion method (inorganic–organic mixed matrix). The common nanomaterial used in NM synthesis is TiOlability . The orgRecently, some authors compared NMs with conventional reverse osmosis membranes at the same pressure, temperature, salinity, and flow rate, resulting in higher productivity (10–20%), constant selectivity (99.5–99.8%), a fouling decrease, and lower energy demand ,85,86 s.MD uses hydrophobic polymeric membranes  of porosities between 0.01 and 0.5 μm. The vapor distillate in MD may be produced by temperature, partial pressure, or vacuum gradients. Direct contact membrane distillation (DCMD), air-gap membrane distillation (AGMD), sweep gas membrane distillation (SGMD), and vacuum membrane distillation (VMD) are different MD configurations that have reached further applications see .2) for thermal gradients between 20 and 40 °C [DCMD and VMD are the most investigated MD configurations . DCMD cond 40 °C ,92,93,94nd 40 °C .Recently, Memsys Water Technologies GmbH  combined the advantages of MED with those of MD processes, resulting in V-MEMD, composed of stages that operate under vacuum with an adequate temperature difference to increase flux through the membrane. In V-MEMD, refrigeration flux in the last stage (highest vacuum) is partly used as preheated feed to the system. The feed passes through each stage, evaporating part way in a cross-flow with the vapor that comes from the thermal system .This emerging technology has been addressed as a sustainable and cost-efficient solution to classical membrane-based separation technologies such as RO and membrane distillation .Water desalination by means of FO processes consists of the osmotic dilution of the draw solution (DS) and freshwater production from the diluted DS ,99,100. The FO membrane performance mainly depends on the DS properties. The ideal DS should provide high osmotic pressure (higher than the feed osmotic pressure), be inexpensive and nontoxic, provide easy recovery of the solute, be stable, and reduce internal concentration polarization .3/d [Recently, FO membrane performance towards desalination was improved by modifying both the DS and FO membranes ,104,105.3/d .3 of desalted water. The RED unit’s assessment results show that SGE-RED is environmentally competitive with other renewable sources such as photovoltaics or wind. The high salinity solution treated with RED has a lower salt concentration and serves as feed solution to the RO unit to reduce the pump work.RED operates in the same way as ED except for the fact that the voltage is applied in reverse about three to four times an hour with an overall water recovery of 97% . The eleSED is a developing technology that purifies water using polarization zones by the concentration of ions in porous media, adjacent to an ion-selective membrane. A SED cell consists of two ion exchange membranes or electrodes between which feedwater flows through a charged porous medium with thin double layers that act as a “leaky membrane” . When a Two processes based on polymeric porous media are currently in development and deserve to be considered: graphene oxide membranes (GMs) and aquaporin-based biomimetic membranes (ABMs). On the other hand, hybrid and integrated systems  are considered emerging.Graphene oxide (GO), among various forms of nanomaterials, provides tremendous opportunities for rational design and tailoring for solar evaporation and film filtration because of its high absorption, porous structure, high chemical stability, hydrophilicity, and excellent anti-fouling properties ,114. To Aquaporins (AQPs) are pore-forming proteins in biological cells. These are composed of a bundle of six transmembrane α-helices embedded in the cell membrane. The amino and carboxyl ends face the inside of the cell, whereas the halves resemble each other, apparently repeating a pattern of nucleotides ,122,123.The major obstacle for ABMs is the scaling up for industry applications, since only small-area membranes have been synthesized due to the highly specialized synthesis techniques. The ABM developed by Zhao et al.  had goodIn recent years, hybrid membrane processes have allowed the achievement of better indicators of the desalination process. For example, Ghaffour et al.  presents3/d incorporate energy recovery devices, e.g., Pelton turbine, turbocharger, and pressure exchanger (PX device). These devices recover energy from the same desalination process.Arafat  defined 3 [The Pelton turbine transforms the rejection pressure into kinetic energy see . The pre3 , i.e., a3 , an SEC The comparison method applied in this research can be used in any geographical context. In this case, the expert panel method was applied to determine the best option  of a desalination process for the current Chilean context.3/d of purified water starting from seawater at 20 °C and salinity of 34,500 ppm. The desalination plant is supposed to be installed in the coastal zone of the Atacama Region (northern Chile) because:There is abundant solar radiation ,134 to jAlvez et al.  reportedFragkou and Budds  argued tSpenceley  informedDue to the high energy demand of desalination techniques, there is a great need for alternatives to reduce the salinity from seawater . In addiThe use of brackish groundwater often brings risks and obligations to an agricultural system. The application of desalinated water for irrigation can promote soil hydrological functions . HoweverThermal and membrane processes were evaluated, aiming at applying them to a pilot scale plant that delivers 100 m(1)Technical characteristics,(2)Scale-up potential,(3)Temperature effect,(4)Electrical supply options,(5)Economic viability.Initially, the classification of information and definition of the technologies to be compared were based on a systematic literature review. Subsequently, the comparative analysis was divided into five categories:Each category was discussed through an expert panel method ,144,145,Incipient: technological development at theoretical and/or laboratory scale.Emerging: technological development prototype at pilot scale.Medium–high: technological development as commercial-scale equipment.High: consolidated technological development and conventional technology, in continuous improvement.Complex: unstable operation.Moderately complex: stable operation.Relatively easy: complicated operation and operation with automation.Easy: very stable process operation and of easy automation.low: the current context does not allow operation with NCRE.medium: the future context does not allow operation with NCRE.high: the current context allows operation with NCRE.demanding: the pretreatment is very necessary to take care of the principal process.moderate: a complex pretreatment is necessary to achieve the goal of the principal process.Simple: a simple pretreatment is necessary to achieve the goal of the principal process.low*: the current context does not allow industrial scaling.medium*: the future context does not allow industrial scaling.high*: the current context allows industrial scaling.The results of this first evaluation allowed us to determine the technologies with the best characteristics to be applied in Chile and thus focus the analysis of the following categories on those.Alternative 1 (A1): Nanofiltration;Alternative 2 (A2): Nanomembranes;Alternative 3 (A3): Forward Osmosis + Reverse Osmosis;Alternative 4 (A4): Solar Distillation.Four alternatives were analyzed in categories 2 and 3, based on the results of category 1:3/d of permeate, with continuous operation for 24 h per day. Low quality: salt content reduced by 50%.Medium quality: salt content reduced by 51–75%.High quality: drinking water quality.Worst: productivity, quality, and cost are lower than RO.Equal: productivity, quality, and cost are similar to RO.Best: productivity, quality, and cost are higher than RO.Worst*: innovation is not better than the current condition.Equal*: innovation equals current condition.Best*: innovation improves the current condition.None: the integration of renewable energy into the desalination process is not possible.Partial: the integration of renewable energy into the desalination process is partially possible.Yes: the integration of renewable energy into the desalination process is possible.In this research, a comparison between the different alternatives was made considering a production level of 100 mFor water quality according to regulations, chloride content is a critical parameter and, if water quality produced is lower than expected, then additional treatment is necessary. The innovations in the operation are the introduction of a new technology to satisfy some need of the desalination process, with science being knowledge, and technology its practice, e.g., seawater feed at a higher temperature.The temperature effect (category 3) was analyzed for thermal and membrane desalination. Membrane desalination was analyzed based on permeate flux density considering the design equations based on the well-known solution diffusion model.(O1) photovoltaic solar plant + electrical network,(O2) photovoltaic solar plant + batteries,(O3) wind turbine + electrical network.The alternative that obtained the best results in categories 1, 2, and 3 was analyzed in category 4 . The analyzed options were:3/d of permeate and consumes 2.09 kWh/m3 (from pretreatment to posttreatment). This plant must have a daily consumption of 188 kWh, i.e., 7.8 kW/h during the 24 h. The results of the present value of cost (PVC)/m3 of water were analyzed considering an internal return rate of 8% and a 20-year horizon.These options were established to ensure an integrated operational system, constant electricity supply, and utilization of NCRE. The electrical supply options were analyzed based on costs associated with a membrane desalination plant that produces 100 mFinally, analysis of category 5 (economic viability) applies to the best result obtained in category 4. The economic viability  and Operating expenses (OPEX)) of the electrical supply option with the lower associated costs was analyzed. We considered a 20-year time horizon. Costs associated with adduction systems, water distribution to customers, and discharge of rejection into the sea were not considered. CAPEX was determined based on the quotations provided by current suppliers in the market.Nowadays, MVC, MED, and MSF contribute 40% of desalinated water worldwide (39 Mton/d) . CurrentThe maintenance cost is similar for all technologies, while the technologies that use lower streams presented lower investment and operating costs. This behavior is in agreement with that reported in the literature ,100.All desalination technologies have the drawback of returning a concentrated solution to the sea; therefore, the minimization of the effect of a local sea salinity increase due to the reject flow is now the technological challenge.Based on the previous results, the technologies chosen by the expert panel to be studied in detail were: NF, NM, FO + RO, and SD. This is because: (1) the technologies supplied with fossil fuel as an energy source were discarded, given the energy context in Northern Chile, (2) SD is an emerging technology with great potential for development in Northern Chile due to greater solar radiation in the area, (3) RO, NF, and NM were the membrane processes that obtained the highest scores, and (4) RO + FO is an emerging process that allows reducing the SEC of traditional RO.Temperature is a variable that influences desalination processes. The SD process, based on phase change, will increase its productivity (evaporation flow) by less than 5% if the operating temperature increases from 20 to 30 °C. In contrast, NM and RO processes, based on the mass transport through semi-permeable dense polymers, will increase their productivity by around 20%, consistent with what the solution diffusion model predicts ,150.The preceding results indicate that the best alternative is NM. O1 and O2 consider the use of solar panels installed near the plant. The photovoltaic solar plant loses 20% of its energy, therefore, 9.8 kWh for 24 h are required. These energy requirements are met all year round for 7 h (10 a.m.–5 p.m.) due to solar radiation in Northern Chile. Instead, O3 considers a 25 kW wind turbine. This equipment delivers the energy requirement to the plant for 5 to 7 h (September to April). The conversion efficiency from wind energy to electrical energy is 25–45%.These results suggest that O1 is the most favorable in the coastal zone of Northern Chile, due to the great potential of solar energy and low investment and maintenance costs.All three options have the seawater desalination system in common, therefore the options share the same economic analysis. 3 of purified water.For the comparative analysis of the three options, the PVC was used, considering: internal rate of return (IRR) equal to 8%, total evaluation period of 20 years, and operating costs, such as: labor, chemicals, clean-in-place (CIP) cleaning, membrane replacement, spare parts, and plant maintenance, resulting in 0.39 USD/m3, USD 0.15/m3, and USD 0.115/m3, respectively. In addition, if the plant operates only with an electrical network, then its PVC is USD 0.42/m3.The relevant difference between the options is the cost associated with the type of electrical energy. In this way, the PVC was calculated considering exclusively the item energy, both in CAPEX and OPEX. This implies that O1, O2, and O3 have a PVC of USD 0.085/m3/d, and a 40 ft container with thermal and acoustic insulation, is 71–74% of the overall cost of the project. When incorporating the cost of a seawater desalination system with a daily production of 100 m3/d and total operating costs, in this case, a PVC of USD 0.81/m3 is obtained, that is, between 32% and 35% lower compared to the conventional reverse osmosis system, for a plant with a capacity of 100 m3/d.The results indicate that the use of NM together with a mixed energy supply system (Alternative 2 + Option 1) is profitable. The cost of a seawater desalination system with a daily production of 100 m3 in the case where only wind and solar potentials are put into practice. Thus, Iran becomes a prototype in the solutions for water scarcity in cases of proper investment and planning. Jimenez [3.These results reinforce similar studies by some authors in other geographic locations. For example, Mollahosseini et al.  analyzed Jimenez  evaluate Jimenez  studied In this research, an innovative operational arrangement was identified within the different seawater desalination technologies that can compete in regard to costs  with conventional RO, through a feasibility evaluation of a pilot plant located in Northern Chile. Therefore, the future implementation of the proposed pilot plant will allow obtaining the experimental data.Currently, membrane desalination processes that use energy recuperators have an SEC 50% lower than thermal desalination processes. NM technology exhibits the most favorable technical characteristics and the best economic indexes to consolidate in the desalination market. Even further, NM represents a competitive alternative versus conventional RO process. A profitable desalination process must consider NM, use of energy recuperators, and mixed energy supply. NM + photovoltaic solar plant + electrical network is the most favorable option in the coastal zone of Northern Chile, due to the great potential of solar energy and low investment and maintenance costs. The incorporation of aquaporins as an emerging process is the emerging lower-cost technology with the greatest potential for seawater desalination. A challenge for all desalination technologies is to minimize the effect of local sea salinity increases due to reject streams. This document presents an up-to-date overview of the impact of emerging technologies on desalinated water quality, process costs, productivity, renewable energy use, and separation efficiency. Said information shows that new desalination technologies are more efficient and competitive. NMs are probably the technology that will be used in the future for desalination processes.


Cervical spondylotic radiculopathy (CSR) is the most common type of cervical spondylosis, accounting for about 60% of the incidence of cervical spondylosis. Both cervical traction and traditional Chinese medicine hot compress are common and effective treatment for CSR. This study will be performed to investigate the effect of a combination of cervical traction and traditional Chinese medicine hot compress on CSR. In this non-blinded, randomized controlled trial, 100 eligible patients will be randomly divided into a treatment group  and a control group . Before and after the intervention, the Visual Analog Scale score, Neck Disability Index score, and 20-score scale of symptoms will be evaluated at baseline and at 7, 14, 21, and 28 days. During the treatment period, any signs of acute adverse events, such as paralysis of aggravated pain, nausea, dizzy, and even syncope, will be recorded at each visit. Although intermittent cervical traction and traditional Chinese medicine hot compress have been used in the treatment of CSR in China for many years, there is no consensus on its effectiveness of combination therapy. This experiment will provide convincing evidence of the efficacy of intermittent cervical traction combined with traditional Chinese medicine hot compress in the treatment of CSR. The treatment methods of CSR are mainly divided into surgical treatment and conservative treatment. Surgical treatment is not considered as the first choice for CSR. At present, there are a lot of nondrug conservative treatment methods, such as acupuncture, massage, Daoyin exercises, traditional Chinese medicine compresses, low-frequency therapy, microwave, etc., and they all have certain treatments for CSR. In the literatures, the clinical efficacy of conservative treatment and surgical treatment is equivalent. Therefore, it is great significant to explore how to use the optimal combination of conservative treatments to improve the neck pain and radicular symptoms of patients with radiculopathy, and alleviate the progress of cervical spondylosis.Cervical spondylosis is a syndrome in which degenerative changes or secondary changes of cervical intervertebral discs stimulate nerve roots, spinal cord, vertebral arteries, or sympathetic nerves, etc., which cause related symptoms and signs.–6:Cervical traction is a common and effective treatment for CSR. Traction relieves the stimulation and compression of nerve roots according to improving the mechanical balance of the cervical spine, changing the relationship between disc protrusions or osteophytes and surrounding tissues. What the functions of intermittent traction are concluded1.The force of intermittent traction is greater than the continuous traction, which is more conducive to stretch the intervertebral space and intervertebral foramen and relieve symptoms.2.Intermittent traction can also cause the neck muscles to have a rhythmic alternating movement of tension and relaxation, resulting in a local massage effect, which is more conducive to relieve neck muscle spasm and eliminate inflammation around the nerve root.3.Intermittent traction can restore muscles and blood vessels, and fewer symptoms such as headaches and nausea after traction, reduce the long-term direct compression of local muscles and blood vessels by the traction belt during traction, and reduce reflex caused by muscle and vasospasm. It is more likely to be accepted by patients.4.In the process of intermittent traction, the muscles are relaxed and combined with work and rest, which reduces the possibility of continuous traction to the neck and back muscles and better relieves muscle spasms. and restores the balance of the dynamic system. And hot compress is with the help of the warming effect to bring the effective ingredients of traditional Chinese medicine into the meridians and collaterals directly to the location of disease, and form a higher concentration in the neck area, fully synergizing the warming and pharmacological effects, so as to achieve the effects of warming the meridian, promoting blood circulation and relieving pain. This project intends to conduct a prospective randomized controlled trial of herbal hot compress combined with traction for the treatment of CSR patients to verify the effectiveness and safety of hot compress combined with traction for CSR treatment.Traditional Chinese medicine hot compress is to directly place the herbal hot pack directly on the neck, exerting the thermal effect, heating up local tissues, expanding capillaries, accelerating blood circulation, improving local metabolism, and achieving the effects of anti-inflammatory, swelling, and reflex muscle spasms. It relaxes the muscles, relieves fatigue and pain,2This study is a prospective randomized controlled clinical trial. The purpose of the proposed study is to investigate the efficacy of traditional Chinese medicine compresses combined with intermittent traction on CSR. The Principal Investigator (PI) is responsible for the entire project and organizes the steering committee meeting. The independent steering committee will be responsible for the safety of participants, meetings, recruitment and follow-up of participants, and quality control. The coordination center is responsible for communicating agreement changes and providing materials. The trial includes a 4-week treatment period and a 4-week follow-up period. After randomization, the patients will receive 2 courses of treatment within 4 weeks. Efficacy evaluations will be performed 7 days after the start of treatment, 14 days after treatment, 21 days after treatment, and 28 days after treatment . All participants will have enough time to make a decision to sign the consent form before the study.,10 This agreement has been registered on Clinical Trials .This study will be conducted in accordance with the principles of the Helsinki Clinical Research Declaration.2.1.4The control group will be treated with intermittent cervical traction combined with hot compresses , and the traction angle will be 10° to 15°; its starting mass will be 3 to 6 kg, according to the condition and the patient's The degree of tolerance can gradually increase, up to 8 to 12 kg; the traction time is 90 seconds, and the intermittent time is 10 seconds; the traction is used for hot compress treatment with an instrument matching hot compress bag, the temperature is controlled at 40 to 50°C, and the neck is applied externally. Once every day, 20 minutes times, a week of treatment for 5 days, 14 days as a course of treatment, a total of 2 courses of treatment; curative effect evaluation on 7 days, 14 days, 21 days, and 28 days after the start of treatment.The intervention group will be treated with intermittent cervical traction combines with traditional Chinese medicine hot compress, and the traction method is the same as the control group. Hot compress herbs include: Corydalis, Tougucao, Frankincense, Myrrh, Angelica, Sichuan pepper, Safflower, Ligusticum chuanxiong, Licorice, Weilingxian, Parsnip, Angelica dahurica, first soak in appropriate amount of warm water and steam it in a steamer for 10 minutes. The temperature of hot pack is controlled at 40 to 50°C, and applies traditional Chinese medicine to the neck. Once a day, 20 minutes times, a week of treatment for 5 days, 14 days as the efficacy evaluation, 7 days, 14 days, 21 days, and 28 days after the start of treatment  software (version 21.0), generating random numbers, and patients will be randomly divided into treatment group (50 cases) and control group (50 cases).2.1.6 and then determine the VAS score by measurement.Pain is one of the most common reasons for patients with CSR to see a doctor. Therefore, the Visual Analog Pain Scale (VAS) will be chosen as the main outcome measure for the assessment of the neck and upper limbs. The VAS score is a reliable and effective method of pain measurement. There is a 100 mm long horizontal line. The left side records “no pain” (score 0), and the right side records “pain is severe and intolerable” (score 10). The patient is required to place a mark corresponding to its current pain level on the horizontal line (the most severe pain at rest and during exercise), is one of the most commonly used indexes to evaluate patients neck dysfunction. The NDI score includes 4 subjective symptoms  and 6 items related to activities of daily living . Each item has 6 levels of responses, each with a score of 0 to 5, 0 means no disability, 5 means severe disability. The lowest total NDI score is 0 points, and the highest is 50 points; the higher the score, the more serious the degree of dysfunction. This study adopted all dimensions of NDI score.Neck Disability Index (NDI)Tanaka Yasuhisa Cervical Spondylopathy Symptom Scale is the “Nerve Root Cervical Spondylopathy Symptom Scale” proposed by the Japanese scholar Hisasu Tanaka. It compares the improvement index before and after treatment for evaluation: improvement rate = (points after treatment-points before treatment)/(20 -Points before treatment)∗100%.2.1.7To ensure the safety of participants, the data collection process will be supervised by the project leader. At each supervision, participants will be required to stay in the hospital for 30 minutes after treatment and asked about any signs of acute adverse reactions during the study. Side effects will be recorded at every visit during treatment.2.22.2.1P < .05 is statistically significant. The mean ± standard deviation will be used to describe continuous variables. This percentage is used to describe the variable. Continuous variables that follow a normal distribution will be analyzed by Student t test; otherwise, nonparametric tests will be used to compare group differences.Trained statisticians will use intentional processing methods for effectiveness and safety analysis. In addition, the final outcome observation index method will be applied to deal with missing values. All statistical analysis will be performed by the SPSS 21.0. The statistical test is two-way, 2.2.2This is a 4-week clinical trial. Participants need to conduct a 4-week research intervention during a 4-week follow-up. Five rounds of disease activity assessment  will be independently recorded by 2 investigators in Epidata (version 3.1), and differences will be resolved through discussion and a third research institute. Longhua Hospital affiliated to Shanghai University of Traditional Chinese Medicine is responsible for monitoring and quality control.2.2.3Before conducting the study, we will conduct unified training to ensure that all relevant doctors, nurses and evaluators fully understand the entire trial process. In order to ensure the quality of the entire study, 2 supervisors will be sent every month to ensure that the enrolled patients meet the inclusion criteria and do not meet the exclusion criteria; the research group recruits enough patients to participate in the study; all enrolled patients fully follow the clinical trial Process, perfect the case report form (CRF), follow the standard operating procedure (SOP). During the entire treatment and follow-up period, the inspector will record the dropout, withdrawal, and related reasons in detail, and record any compliance of all patients.3 At the same time, it activates a variety of cell signal transduction pathways, cytokines and receptor proteins in the body, which in turn can induce interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor- α (TNF-α) and other pro-inflammatory cytokines are secreted to mediate inflammation and form a vicious circle of inflammation. The heat generated by herb compresses causes blood vessels to dilate and muscles relax. Heat therapy is a common alternative therapy to improve blood circulation and local muscle spasms. The various ingredients of herb have a variety of analgesic and anti-inflammatory properties. The herb in this research contain Corydalis, Yang Bin, etc. When studying the extracts of Chinese medicine Corydalis Corydalis, they found that Corydalis extracts can exert the same effect as NLRP3 inhibitors. Inhibit NLRP3 inflammasome signaling pathway protein expression. The NLRP3 inflammasome signaling pathway can be widely involved in the body's inflammatory response and promote the synthesis and secretion of inflammatory factors such as prostaglandin E2 (PGE2), phospholipase A2 (PLA2), IL-6, and TNF-α. Corydalis extract inhibits NALP3 and then inhibits the inflammatory response, relieves the inflammatory response caused by nerve root compression, and reduces secondary spinal cord injury.The clinical features of CSR are that the pain range is consistent with the compressed spinal nerve area, mainly pain, numbness, and hypoesthesia. In severe cases, radicular weakness, and muscle atrophy may occur. After the occurrence of CSR, a large number of inflammatory factors are released from the damaged tissue, which stimulates the nerve root and causes radicular pain symptoms. and relieve it quickly. The first symptoms of CSR are contradictory. The active ingredient xanthamide extracted from Zanthoxylum bungeanum can significantly increase the pain threshold of the CSR body, so that the body can withstand greater intensity of pain stimulation. Moreover, prickly amide can reduce the levels of inflammatory factors TNF-α, IL-1β, and cyclooxygenase-2 (COX-2), play an anti-inflammatory and analgesic effect, and have a certain control effect on the symptoms of CSR [24]. The active ingredient ferulic acid (FA) extracted from Ligusticum chuanxiong can also increase the pain threshold of the CSR body, and Zhao Weihong and others have observed that FA can significantly improve the obvious edema and degeneration of nerve root tissue and demyelination caused by lesions. To improve the potential motor conduction velocity (MCV) of the median nerve of the CSR body.Traditional Chinese medicine can not only inhibit nerve root inflammation, relieve nerve root edema, and fundamentally alleviate the root symptoms of CSR, but also can increase the body's pain threshold, improve the body's spontaneous pain and hyperalgesia caused by inflammatory stimulation,Therefore, we plan to conduct this prospective randomized controlled clinical trial to study the clinical efficacy of traditional Chinese medicine hot compress combined with traction in the treatment of cervical spondylotic radiculopathy, and to explore how to use the most combination of conservative therapies to improve cervical pain and cervical radiculopathy. Radical symptoms and explore whether it can be the first choice for potential conservative treatment of CSR.Xing Ding, Jinze Wu are co-first authors of this manuscript, contributing equally to the design, conduct of the trials, and drafting of the manuscript. All authors participated in the design of the study and performed the trial. Jinhai Xu and Wen Mo are the co-corresponding authors of this manuscript, contributing equally to the supervision and coordination of the clinical trial. All authors read and approved the final manuscript.Conceptualization: Xing Ding, Jinze Wu, Qixing Shen, Wen Mo.Data curation: Xing Ding, Jinze Wu, Jinhai Xu, Wen Mo.Formal analysis: Xing Ding, Jinze Wu, Qixing Shen, Jinhai Xu, Wen Mo.Funding acquisition: Xing Ding, Jinze Wu, Qixing Shen, Jinhai Xu, Wen Mo.Investigation: Xing Ding, Jinze Wu, Qixing Shen, Jinhai Xu.Methodology: Xing Ding, Jinze Wu, Qixing Shen.Project administration: Xing Ding, Qixing Shen, Jinhai Xu, Wen Mo.Resources: Xing Ding, Qixing Shen, Jinhai Xu, Wen Mo.Software: Xing Ding, Jinze Wu, Jinhai Xu, Wen Mo.Supervision: Xing Ding.Validation: Xing Ding, Jinhai Xu, Wen Mo.Visualization: Xing Ding, Jinze Wu, Jinhai Xu.Writing – original draft: Xing Ding, Jinze Wu, Jinhai Xu, Wen Mo.Writing – review & editing: Xing Ding, Jinze Wu.


Recently, aptamers have attracted attention in the biosensing field as signal recognition elements because of their high binding affinity toward specific targets such as proteins, cells, small molecules, and even metal ions, antibodies for which are difficult to obtain. Aptamers are single oligonucleotides generated by in vitro selection mechanisms via the systematic evolution of ligand exponential enrichment (SELEX) process. In addition to their high binding affinity, aptamers can be easily functionalized and engineered, providing several signaling modes such as colorimetric, fluorometric, and electrochemical, in what are known as aptasensors. In this review, recent advances in aptasensors as powerful biosensor probes that could be used in different fields, including environmental monitoring, clinical diagnosis, and drug monitoring, are described. Advances in aptamer-based colorimetric, fluorometric, and electrochemical aptasensing with their advantages and disadvantages are summarized and critically discussed. Additionally, future prospects are pointed out to facilitate the development of aptasensor technology for different targets. Owing to the advances in various areas of life, the population has become more exposed to pollutants, whether from industry, agriculture, or medical waste, resulting in an increase in the pollution associated with humans’ daily lives, leading to an increase in the rate of diseases. In this context, the development of fast, simple, low-cost, high-sensitivity, and specific sensors for detecting pollutants or early stages of diseases is important.Because of the great advances in molecular biology and genetic engineering, the use of RNA and DNA has expanded not only in biology, for storing and transmitting genetic information, but also in the identification of antibiotics, proteins, peptides, amino acids, and even small molecules. Gold et al. and Szostak et al. reported the first studies involving a specific targeting affinity toward proteins ,2.Aptamers consist of 3D-folded structures of single-stranded oligonucleotides with lengths of usually 20–60 bases of nucleotides selected in vitro via the systematic evolution of ligand exponential enrichment (SELEX) process. The SELEX process is applicable for single targets, complex target structures, or even mixtures without proper knowledge of their composition. SELEX can be used to select aptamers with high affinities and specificities for their targets and low dissociation-constant values, across the low nanomolar to picomolar range. Aptamers can be selected through the in vitro process independent of animals or cell lines. The selection of aptamers for toxic target molecules or molecules with no or low immunogenicity is possible. Different modifications can be introduced in the basic SELEX process for the selection of the desired aptamer specifications for a specific application. The aptamers can be functional for native conformations of target molecules on live cells, so cell surface transmembrane proteins can be considered as targets ,5,6.The 3D folded structure permits the formation of stable complexes with various targets, such as proteins, nucleic acids, and small molecules ,11,12,13Because of their size-dependent physical and chemical properties, biocompatibility, electronic properties, magnetic properties, and ability to manifest biological signaling and transduction mechanisms, the nanomaterials are providing great advances in the field of sensors. Several nanomaterials have been synthesized with different sizes and shapes such as gold nanoparticles (AuNPs), silica nanoparticles, silver nanoparticles (AgNPs), magnetic nanoparticles (MNPs), and carbon quantum dots (CQDs) as unique templates for many applications such as biomaterial assays; the diagnosis, monitoring, and treatment of disease; and drug delivery ,36,37,38This systematic review is focused on recent approaches to aptamer engineering for biorecognition for several objectives. Herein, previous and current advances related to aptamer-based sensing protocols are provided, highlighting the possible detected signals, with a focus on the use of different nanomaterials with distinct configurations. The most significant studies on aptasensor development have been collected, providing the possible strategies available for using aptasensors. Moreover, a deep discussion on colorimetric, fluorescence, and electrochemical detection strategies is provided.Among the available assay recognition signals, colorimetric methods are considered simple and efficient with a great potential for point-of-care diagnostics, as the detection responses are simply visually discerned by the naked eye using simple, low-cost, and effective instrumental techniques. The basic colorimetric strategies involve the detection of the target by a color change through the naked eye and simple instrumentation. Colorimetric biosensors based on aptamers have demonstrated their sensitivity and selectivity, in addition to their effective potential for rapid onsite diagnosis without complicated instrumentation. However, the colorimetric aptasensor has some limitations such as the influence of color from samples; the time-consuming nature of the fabrication process ,58; the Nobel metal nanomaterials, in particular, AuNPs and AgNPs, are excellent signal transducers for colorimetric analysis due to their significant optical properties associated with their particle size, size distribution, and shape ,62,63. TEscherichia coli (E. coli) assay based on AuNPs’ aggregation as described in E. coli, establishing a stabilizing layer around the AuNPs. In the presence of the E. coli target, an aptamer–E. coli competitive interaction with AuNPs occurs, inducing the aggregation of AuNPs, with a distinct color change from red to blue and a low limit of detection (LOD) of 101 cells/mL, which is observed visually by the naked eye . S.. S.Eschey Jiecan . The AuNd et al. . In thisd et al. . Similard et al. , as show2+, Salmonella typhimurium, and AFB1, changing the specific aptamer functionalized with the AuNPs [As previously discussed, several colorimetric aptasensors were developed based on AuNP-induced aggregation with different targets such as malathion, Cdhe AuNPs ,87,88,90+) was also evaluated using their specific aptamers. The study aimed to examine if the binding between the target and AuNPs  could affect the analytical reliability of the sensor. Liu et al. claimed two possible mechanisms, in which a aptamer specific for dopamine could adsorb on the surface of AuNP as described in +3, as shown in Recently, Zong et al., and Liu et al. ,92 propoSalmonella typhimurium (S. typhimurium) based on S. typhimurium’s affinity, to enhance the peroxidase-like activity of bovine serum albumin-stabilized gold nanoclusters modified with dual aptamers (aptamers@BSA-AuNCs), as outlined in S. typhimurium, the aptamers@BSA-AuNCs could oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB), producing the blue oxidized form, with a wide linear response to S. typhimurium in the concentration range of 101–106 CFU/mL and LOD of 1 CFU/mL [The AuNPs were confined for using in a colorimetric aptasensor with different strategies other than aggregation, depending on the catalytic performance of the NPs and anisotropic growth of the AuNPs. Chen et al. proposed a colorimetric aptasensor for the selective and sensitive detection of 1 CFU/mL .4 and NH2OH as reducing agents, a uniform or anisotropic growth of AuNPs with a distinct change in the localized surface plasmon resonance (LSPR) could occur based on the amount of bound aptamer on the surface of the bare seed AuNPs, which is related to the concentration of ABA, with a linear relationship for 1 nM to 10 μM and LOD of 0.51 nM.Wang et al. proposed a colorimetric strategy for abscisic acid (ABA) detection based on the anisotropic growth of AuNPs in the presence of aptamer on the surface of the seed AuNPs . In the Furthermore, Seongjae Jo et al. and Wonjung Lee et al. ,100 desc2+ assay assisted by a specific Cd2+ aptamer based on the enzyme-mimicking activity of MoS2 nanocomposites, as reported by Tao et al. [2+ aptamer was immobilized on the bottom of the microplate via biotin–avidin affinity; simultaneously, a functionalized complementary strand CsDNA–Au–MoS2 was used as a signal transducer, depending on its peroxidase-like activity toward the chromogenic TMB. This system exhibited a linear range of 1–500 ng/mL and a LOD of 0.7 ng/mL.A colorimetric strategy other than AuNP aggregation was developed for a Cdo et al.   biomarkers colorimetrically, as provided by Li et al. in 3O4 nanoparticles in the conversion of the colorless TMB into blue TMB was enhanced significantly after functionalization with an ATP aptamer. In the presence of an ATP target, the target binds with its aptamer, which departs from the surface of the Fe3O4, decreasing its activity, with a detection range of 0.50–100 μM and a LOD of 0.09 μM. The toxic Pb2+ was also assayed via a colorimetric aptasensor based on the peroxidase-like activity of graphene/Fe3O4-AuNP composites, showing a linear range of 1–300 ng/mL and a LOD of 0.63 ng/mL, as reported by Tao et al. [Magnetic nanoparticles (MNPs) have also been used as a nanoassembling template for bioassay targeting, either alone or in combination with other nanostructures, where they can be easily isolated from the solution via an external magnetic field ,104,105.o et al.  , as reported by Duan et al.’s group [E. coli bacteria. The detection aptamer was fabricated with Cu-MOF NPs through avidin–biotin affinity as a transducer signaling based on the peroxidase activity of the Cu-MOF NPs catalyzing the conversion of the colorless TMB into the oxidized blue structure and, then, yellow in the presence of the acid. This modified ELISA succeeded in detecting 16 to 1.6 × 106 CFU/mL, with a LOD of 2 CFU/mL [β-estradiol-E2 (17β-E2) by Huang et al. [3O4 NP–graphene oxide composite was functionalized with complementary DNA and served as the signal producer for assaying the PSA. In the absence of PSA, the Fe3O4 NP–graphene oxide–DNA probes were captured by the PSA aptamer; therefore, the Fe3O4 NPs were transformed into Prussian blue (PB) using a HCl solution, which was then mixed with potassium ferricyanide to produce a multicolor signal; simultaneously, the PB can produce a thermal signal upon near-infrared laser irradiation, with LODs of 0.15 and 0.31 ng/mL for colorimetric and photothermal assays.Replacing the antibodies with aptamers in the ELISA protocol has attracted attention for assaying food-borne pathogens  for the rapid assay of  O157:H7 , as descFluorescence-based aptasensors are characterized by high sensitivity, large-detection ranges, multiplexing capabilities, rapid assaying, and the highly selective recognition of aptamers for several targets, which can be distinguished under UV light, in comparison with the colorimetric sensor’s signals that are distinguished using visible light. The integration of both fluorescent materials (fluorophore dyes and fluorescent nanoparticles such as upconversion nanoparticles (UCNPs), GO, and CQDs) and aptamers can produce high sensitivity and selectivity, and a rapid analysis strategy, making them useful candidates for fluorescence-aptasensor bioassays ,114,115.The design of fluorescent aptasensors requires the use of hairpin aptamers (aptabeacons), which are labeled with either a fluorophore or a quencher. Forster resonance energy transfer (FRET) typically uses a donor fluorophore and an acceptor quencher material. Different strategies are often employed via FRET operations, which are performed through either “signal-on” or “signal-off” assaying protocols. These operations are based on the disparity in the fluorescence responses of the fluorophores as a function of the potential and unique aptamer–target binding and the conformational-change degree ,117,118.Some of the relevant fluorescent strategies were employed for the assaying of several targets based on integrated aptamers and fluorescent materials as described in the following Aspergillus, Penicillium, and Byssochlamys, be assayed via fluorometric aptasensors . This MOF can quench the fluorescence of the TAMRA with a sensitivity of 0–180 ng/mL and a LOD of 0.35 ng/mL. 3,4,9,10-perylenetetracarboxylic acid diimide (PTCDI), an affordable and low-cost fluorophore dye, was used for the assaying of ampicillin (AMP). This assaying was based on the competitive quenching between the AMP aptamer, and the complementary strand (CS) and AuNPs . In the presence of thrombin or ATP, the respective aptamer binds with its target, leading to the restoration of the fluorescence, with LODs of 1.2 and 13 nM for thrombin and ATP, respectively . The aptnd AuNPs F, with aγ). As outlined in A method for signal amplification was introduced by Wen et al., who applied dual atom transfer radical polymerization (ATRP) technology for amplifying the fluorogenic signal during the assaying of gamma-interferon protein (IFN-3O4@GO as a single-stranded DNA (ssDNA) adsorbent for the detection of isocarbophos [Polydiacetylene (PDA) liposomes were immobilized with an aptamer-tagged fluorophore (Cy3–Apt) for assaying mucin 1 (MUC1), as indicated in the proposed scheme shown in arbophos   are considered perfect nanoquenchers for fluorophore-labeled ssDNA, owing to the stacking of nucleotides on the surface of the multiwalled carbon nanotubes (MWCNTs), which induces quenching via the FRET system. The hybridization of the aptamer with the target leads to the restoration of the fluorescence. Therefore, the ssDNA–MWCNT assembled fluorescence probe was used for the assaying of Hg2+ .E. coli bacteria has been developed based on the FRET between aptamer-functionalized UCNPs as donors and layered tungsten disulfide (WS2) as the acceptor. This was achieved through the attachment of the aptamer to the WS2 surface via van der Waals forces. In the presence of E. coli, the specific aptamer binds preferentially to E. coli, causing a conformation of the aptamer that induces the dissociation of the probe away from the surface of the WS2 nanosheets. Therefore, part of the fluorescent intensity is restored as a function of E. coli , with modified AuNPs, with a detection range of 25–1400 nM and LOD of 5.7 nM [Rare-earth-element-doped UCNPs can convert low-energy light into high-energy light with short wavelengths via the photon mechanism. UCNP materials are characterized by high quantum yields, narrow emission peaks, excellent photostability, long fluorescence lifetimes, environmental friendliness, low toxicity, bulky anti-Stokes shifts, and high resistance to photobleaching. Moreover, infrared light induces minimal photodamage to the biological targets without exciting the biological fluorophoric substance, which can be confined in the matrix ,138,139. E. coli A, with c7 CFU/mL . Moreove0 CFU/mL . A simil0 CFU/mL . Pb2+ waf 5.7 nM . Basicalf 5.7 nM . Fluoresf 5.7 nM . Qiu et f 5.7 nM . Once thf 5.7 nM .As mentioned above, aptamers are sequences of nucleic acid, and because of that, they can be integrated with a DNA system for fluorescent signal amplification to improve the detection limit and, therefore, be used in the early diagnosis of diseases. These amplification processes include rolling-cycle amplification, strand displacement reactions, hybridization chain reactions, and hairpin DNA cascade hybridization reactions ,150,151.Aggregation-induced emission (AIE) was conducted for assaying exosomal tumor-associated proteins using GO as a quencher and TPE-TA (tertiary amine-containing tetraphenylethene) as a fluorescent dye C. The po2+ in environmental and agricultural samples with the assistance of a specific aptamer. The MIP was integrated with aptamers to make dual recognition units, while carbon-doped sulfur, nitrogen quantum dots, and AuNPs (SN-CQD/Au) were used as a fluorophore. The fluorescence was quenched via Cd2+, with a linear response range of 20 pM–12 nM and a LOD of 1.2 pM. [The aptamers themselves can be modified with a fluorophore and quencher materials at both ends without affecting their binding affinity toward the targets, so a FRET aptasensor strategy can be easily constructed in the presence of targets inducing fluorescence quenching ,157,158. 1.2 pM. .Zhang et al. developed a simple, sensitive, and label-free method to assay the cyanotoxin microcystin–leucine–arginine (MC-LR), using fluorescent dsDNA–CuNCs as a fluorescent probe, comprising an aptamer for MC-LR and its complementary ssDNA. The presence of the MC-LR target results in the uptake of the aptamer from the dsDNA–CuNCs; therefore, the fluorescent signal exhibits a sensitivity range of 0.01 to 1000 μg/L and LOD of 4.8 ng/L . Sharma An electrochemical aptasensor was fabricated using an aptamer as a bioreceptor and an electrochemical transducer, which translated the target–aptamer affinity into a measurable electrochemical signal through potentiometry, voltammetry, amperometry, impedimetry, or electrochemiluminescence. The potentiometric approach involves the measurement of the potential between the probe and the reference electrode without any net charge transfer. The amperometric approach is based on applying a potential and allowing a redox reaction to occur. The signal is defined as the current between the electrode and the counter-electrode. The voltammetric strategy includes the sweeping potential over time and recording the corresponding current; this strategy is based on a three-electrode system, with working, reference, and counter electrodes. The impedimetric technique involves measuring the charge transfer rate on the surface of the electrode for a kinetic study.The electrochemical aptasensors were modified with various nanomaterials such as carbon-based nanomaterials, metal–organic frameworks (MOFs), AuNPs, and polymers for signal amplification ,166,167.2+, lysozymes, insulin, thrombin, CD44, vanillin, circulating human MDA-MB-231 breast cancer cells, bisphenol A, furaneol, and Hg2+, among others [The electrochemical aptasensor mainly depends on the interactions occurring on the surface of transducer as a result of the induced reaction between the target and its specific aptamer, providing amperometric or potentiometric electrochemical signals. Another technique is based on the increase in charge transfer resistance via the impedance technique . Therefog others ,188,189.−5 to 10 nM, and a LOD of 3.3 × 10−3 pM [2-g-C3N4 and AuNPs (The chratoxin A (OTA) toxin was detected electrochemically, specifically using the OTA aptamer as demonstrated in  10−3 pM . Amoxicind AuNPs B. BecausA sandwich-type electrochemical aptasensor has been fabricated for a thrombin assay, as demonstrated in 2+, among others—by monitoring the electrochemical signals of the released labeling substrates as a result of competitive targets [−13–1 × 10−5 g/mL and a LOD of 3.1 × 10−14 g/mL. The same technology was also used for assaying PAT; MOF@Methylene blue was used as the signal tag released from the electrode surface as a function of the PAT concentration, with a linear range of 5 × 10−8–5 × 10−1 μg/mL and a LOD of 1.46 × 10−8 μg/mL [2+ was detected with a range of 0.5–25 ppb and a LOD of 0.6 ppb, using toluidine blue as a redox-released tag signal liberated in the presence of Pb2+ [–4 HAU (hemagglutination units) in a 200 μL sample [Recently, enzymes such as HRP, glucose oxidase, and alkaline phosphatase, and electroactive compounds such as QDs, ferrocene (Fc), ferrocyanide, methylene blue (MB), and Cd nanoparticles were successfully incorporated in electrochemical aptasensor technologies to be used as signal enhancers ,194. A d targets ,199,200. targets , as outl−8 μg/mL , as show of Pb2+ . Enzymat of Pb2+ ,202. Fu  of Pb2+ . MagnetiL sample .The electrochemical method was integrated with amplification strategies for assaying numerous targets, such as sulfadimethoxine, malathion, OTA, kanamycin, ampicillin, atrazine (ATZ), and acetamiprid ,209,210.The distinctive and impressive advantages of aptamers compared with antibodies permit them to be preferred in molecular diagnostics for a wide range of biomarkers. Aptamers have intrinsic advantages, such as their availability for both chemical modifications and conjugation with different labels, facilitating their ability to be used to construct a sensitive and highly selective platform sensor. In this review, recent advances in the different methods of employing aptasensors including colorimetric, fluorometric, and electrochemical strategies were discussed. The simplicity and small sizes of the aptamers combined with the versatile optical properties and large surface areas of nanomaterials leads to a platform with great potential for highly sensitive and selective biological recognition and signal transduction for various analytes such as metals, small molecules, toxins, proteins, cells, and bacteria with lower LODs and high sensitivity and selectivity.In this review, each detection method with its own different strategies was emphasized briefly with schematic designs and all the aptamer information, including the detection ranges of the discussed aptasensors, summarized in Biomarker detection based on aptamer functionalization still needs to overcome these limitations in order to be available for the multi-detection of metal ions, DNA, and proteins. Furthermore, the development of more specific aptamers is still needed, as is also integration into sensor platforms. Aptasensor platforms need more attention regarding (1) simultaneous multiple marker detection; (2) the long-term stability of biosensor assays; (3) direct assays in real sample matrixes; (4) understanding the nature of the binding competition between an aptamer and target on the surface of a nanomaterial, which could affect the sensor reliability; (5) more focus on the future development of in vivo aptamer sensing technology, the possible problems, and their solutions; and (6) more intensive research regarding the improvement of the POCT of biomarkers using aptasensors. The aforementioned hurdles need to be overcome quickly for the achievement of a reliable and selective detection of markers with ultrasensitivity, with affordable and portable on-site analytical devices. We are sure that the scientific community has the talent to offer solutions to these hurdles in order to design an affordable, easy-to-use nanomaterial-based electro-optical aptasensor integrated with rolling-cycle amplification technology.


In this study, three-dimensional (3D) biopolymeric scaffolds made from collagen, silk fibroin and chitosan were successfully prepared by the freeze drying method. Dialdehyde starch (DAS) was used as a cross-linking agent for the materials. The properties of the materials were studied using density and porosity measurements, scanning electron microscope (SEM) imaging, swelling and moisture content measurements. Additionally, cytocompatibility of the materials in contact with MG-63 osteoblast-like cells was tested by live/dead staining and resazurin reduction assay on days 1, 3 and 7. It was found that new 3D materials made from collagen/silk fibroin/chitosan binary or ternary mixtures are hydrophilic with a high swelling ability (swelling rate in the range of 1680–1900%). Cross-linking of such biopolymeric materials with DAS increased swelling rate up to about 2100%, reduced porosity from 96–97% to 91–93%, and also decreased density and moisture content of the materials. Interestingly, presence of DAS did not influence the microstructure of the scaffolds as compared to non-cross-linked samples as shown by SEM. All the tested samples were found to be cytocompatible and supported adhesion and growth of MG-63 cells as shown by live–dead staining and metabolic activity test. Bone tissue plays many important functions in the human body, e.g., supporting, transferring load, protecting organs, acting as a reservoir of vital elements and cells . TreatmeA number of protein polymers  and polysaccharides  are used in biomaterials production ,12,13,14Significantly better physio-chemical properties have been demonstrated for silk fibroin ,22. SilkNowadays, many synthetic cross-linking agents including formaldehyde, glutaraldehyde, polyepoxy compound, and hexamethylene-1,6-diaminocarboxysulphonate (HDACS), are used for biomaterial modifications ,32,33. NDialdehyde starch (DAS) has been used for cross-linking of the scaffolds for tissue engineering . DAS is The aim of this work was to design new materials of silk fibroin, collagen and chitosan and to obtain polymeric three-dimensional (3D) matrices based on mixtures of these polymers. Two-component collagen and chitosan mixtures with organic and inorganic additives have been described previously . Binary In this study, three-component materials based on collagen, silk fibroin and chitosan were obtained and their porosity, density, morphology, swelling ability and moisture content were studied. The influence of DAS as a cross-linking agent on properties of the obtained scaffolds was investigated. Biological tests were carried out to study the effect of the obtained materials on osteoblast-like cells.6 g/mol.Chitosan (CTS) was supplied by Sigma-Aldrich . The deacetylation degree  of chitosan was 78%, and the viscosity average molecular weight was 0.59 × 10Collagen (Coll) was extracted in our laboratory, from rat tail tendons ,25. The Bombyx mori cocoons . Cocoons were boiled twice for 1 h in aqueous solution of 0.5% Na2CO3, according to the procedure found in the literature ,3],3],Porosity and density were measured using liquid displacement method with isopropanol as the liquid, because it did not dissolve the samples . Porous Samples of each kind were measured in triplicate.The microstructure of the samples was studied using Scanning Electron Microscope (SEM) . The scaffolds were frozen in liquid nitrogen for 3 min. Freezing of a sample allows its gentle cutting with a razor scalpel to observe its interior microstructure. The samples were sputter-coated with a layer of gold prior to observation. The size of the pores was measured on SEM images using the Sigma Scan Pro 5 software ,46,47. TThe swelling ratio was measured by immersing the composites’ fragments in phosphate buffered saline (PBS) solution, pH = 7.4. After 1 h of immersion, the materials were gently dried by putting them between two sheets of paper, and then weighed . The sweScaffold moisture contents were determined by drying samples in an oven at 105 °C until they reached a constant weight. The results were expressed as grams of water in 100 g of dry sample weight. Samples of each kind were measured in triplicate.2. Prior to cell culture, the scaffolds  were sterilized by soaking in 70% ethanol aqueous solution and washed five times with sterile PBS . The scaffolds were then transferred to 24-well plates and seeded with 1 × 104 cells per sample (suspended in 1 mL of EMEM). The cells seeded directly on tissue culture polystyrene  served as control. The experiment was run in triplicate.Cytocompatibility of the materials was tested using MG-63 osteoblast-like cells . The cells were cultured in Eagle’s minimal essential medium  supplemented with 10% foetal bovine serum, 1% penicillin-streptomycin and 0.1% sodium pyruvate . The cells were cultured at 37 °C, in a humidified atmosphere containing 5% COex = 544 nm, λem = 590 nm, FluoStar Omega, BMG Labtech, Ortenberg, Germany). The percentage of resazurin reduction was calculated according to the following Formula (4):After 1, 3 and 7 days, the viability of the cells was determined using resazurin reduction assay. Cell culture medium was carefully removed from the wells and replaced with 1 mL of fresh cell culture medium containing 5% of AlamarBlure reagent . After 3 h of incubation, 100 µL of the medium was transferred into a black 96-well plate for fluorescence measurement  and 0.1% propidium iodide (Sigma-Aldrich) dissolved in PBS for 20 min at 37 °C. Fluorescence microscopy images were taken using Axiovert 40  microscope with HXP 120 C Metal Halide Illuminator .Porosity and density are very important parameters, which have to be defined for porous scaffolds for tissue engineering. The porosity and density were measured for single polymers  and their mixtures with the addition of chitosan and a cross-linking agent (DAS). The results are presented in Decrease in material porosity upon cross-linking with DAS was accompanied by an increase in sample density in all samples, except Coll, in which density, like porosity, was not affected by the presence of DAS. The most pronounced differences in both porosity and density were observed for SF, which may indicate strong affinity of DAS to SF. The concentration of SF in SF/Coll and SF/Coll/CTS mixtures was lower, thus the influence of DAS was less distinct. Increasing density of material would favour cell growth and proliferation, as more materials can be accessed by the cells ,51. IncrThe morphology of the scaffolds was evaluated with scanning electron microscopy . Each scThe materials made of collagen, silk fibroin and chitosan were easily wettable by polar solvents such as PBS. They exhibited a high swelling ability. It is because collagen, chitosan and silk fibroin contain a large number of functional groups capable of binding water ,61,62. TNo result for collagen can be found in Developed scaffolds are intended to be implanted into small bone tissue defects. For that, the scaffolds have to be non-toxic and facilitate bone cell adhesion and proliferation. To assess biological properties of the developed materials, they were seeded with osteoblast-like MG-63 cells [The results of the biological test were shown only for three types of the samples, i.e., those cross-linked with DAS, because the samples without cross-linking agents were dissolved after 1 day of cell culture. Also, the SF scaffold was dissolved in cell culture medium. Therefore, the positive effect of DAS on the stability of the samples made exclusively of collagen, as well as ones made of binary and ternary blend materials in cell culture medium can be clearly observed. Metabolic activity of MG-63 cells was measured after 1, 3 and 7 days . RegardlThe results of metabolic activity were further confirmed by a live/dead assay. At the beginning of the culture, the number of cells visible on the samples was low, but it gradually increased as the culture time increased. At all time points, live cells were visible on the samples. The distribution of the cells was uniform and migration of some cells into deeper parts of the scaffolds was observed. After 7 days of culture, their morphology was similar to those of the cells cultured on control TCPS. The cells were characterized by an elongated, spindle shape. In the case of the Coll /DAS sample, material degradation occurred very quickly. After 3 days of culture, small fragments of the material detached from the sample very easily, and the sample itself, after being removed from the culture well, was collapsed. Slower, but still visible material degradation also occurred in the other two samples. For this reason, it is difficult to assess whether the observed differences in the metabolic activity of the cells cultured on the tested materials resulted directly from the cytotoxicity of a given material, or whether it was the result of a decrease in the volume or surface of the sample available to the cells.All in all, the analysed samples were cytocompatible with MG-63 cells. Cells grown on these materials proliferated normally and showed morphology similar to control cells. The number of cells growing on the material increased with time and after 7 days of culture, the cells evenly covered almost all the available surface of the material. Our results are consistent with previous studies pointing out that the addition of DAS to chitosan/gelatin scaffolds supported SaOs-2 cell attachment and proliferation without causing any cytotoxicity effects . In otheThe use of other commonly applied chemical cross-linking agents, such as formaldehyde, glutaraldehyde, or glyceraldehyde is limited due to their possible toxicity and leakage of unreacted cross-linker molecules in vivo. Thus, novel and safer cross-linking compounds e.g., genipin are being extensively investigated . Our resThree-dimensional scaffolds based on silk fibroin, collagen and chitosan, cross-linked by dialdehyde starch were obtained by a freeze drying method. They were characterized by high swelling degree, high porosity (about 90%) and interconnected pores less than 200 µm in size. The addition of a cross-linking agent (DAS) modified physico-chemical properties of the materials. These materials had better swelling ability, which is important for tissue engineering applications. All the studied materials were cytocompatible with MG-63 cells. It can be assumed that the scaffolds based on silk fibroin, collagen and chitosan, cross-linked by DAS can be proposed as potential biocompatible matrices for tissue engineering purposes. It is expected that they will promote bone healing and regeneration.


Psychological distress imposed by the SARS-CoV-2 outbreak particularly affects patients with pre-existing medical conditions, and the progression of their diseases. Patients who fail to keep scheduled medical appointments experience a negative impact on care. The aim of this study is to investigate the psychosocial factors contributing to the cancellation of medical appointments during the pandemic by patients with pre-existing health conditions. Data were collected in eleven Italian hospitals during the last week of lockdown, and one month later. In order to assess the emotional impact of the SARS-CoV-2 outbreak and the subject’s degree of psychological flexibility, we developed an ad hoc questionnaire (ImpACT), referring to the Acceptance and Commitment Therapy (ACT) model. The Impact of Event Scale-Revised (IES-R), the Depression, Anxiety and Stress Scale (DASS) and the Cognitive Fusion Questionnaire (CFQ) were also used. Pervasive dysfunctional use of experiential avoidance behaviours , feelings of loneliness and high post-traumatic stress scores were found to correlate with the fear of COVID-19, increasing the likelihood of cancelling medical appointments. Responding promptly to the information and psychological needs of patients who cancel medical appointments can have positive effects in terms of psychological and physical health. On 21 February 2020, the first case of COVID-19, as caused by the SARS-CoV-2 virus was confirmed in Italy [Hospitals in northern Italy were quickly swamped with high numbers of COVID-19 patients. Facilities and resources had to be hurriedly reorganised, as a matter of urgency, so that dedicated COVID-19 units could be opened. At the end of April 2020, 17.4% of all identified individuals with SARS-CoV-2 were experiencing severe symptoms of COVID-19 that required hospitalisation. Meanwhile, 13.6% of individuals with SARS-CoV-2 were asymptomatic, 17.2% pauci-symptomatic, and 35.7% displayed only mild symptoms. The remaining 16.3% had symptoms for which the severity level was not specified. In addition, of all subjects with COVID-19, 1.9% were hospitalised in critical condition requiring intensive care [Comorbidity appears to be a determining factor in COVID-19 severity and prognosis. In 34.7% of cases, at least one co-morbidity has been reported, including cardiovascular diseases, respiratory diseases, hypertension, diabetes, immunodeficiencies, metabolic diseases, cancers, obesity, kidney diseases, and other chronic diseases [The psychological distress imposed by the SARS-CoV-2 outbreak has been widely reported. This particularly affects patients with pre-existing medical conditions, for example, a large proportion of young cancer patients have expressed their deep concern at being more susceptible to severe complications and feeling the burden of their parents’ worry . RheumatIn many cases, patients have been contacted by medical centres to cancel or postpone scheduled consultations, treatments and/or surgery to reduce the risk of contracting SARS-CoV-2. In turn, patients have also contacted medical centres to cancel appointments.It should be noted that many patients have not cancelled their scheduled medical appointments, despite the impact of the external context, including the many social media information (sometimes very conflicting or confusing) and institutions .During the SARS-CoV-2 outbreak, patients’ reasons for cancelled appointments included being unable to: get time off work, secure childcare, and/or find a safe mode of transport. In addition, patients reported cancelling appointments because their health concern had resolved itself, or because they were too ill to attend .Research conducted prior to the SARS-CoV-2 outbreak indicates that patients who schedule medical appointments and fail to keep them have a negative impact on patient care ,12,13. PThus far, there have been no published studies on the behaviour of cancelling medical appointments of patients with pre-existing medical condition during the SARS-CoV-2 outbreak, and especially none that consider the psychosocial factors influencing the decision to cancel appointments regularly scheduled by medical centres.The behaviour of cancellation medical appointments can be linked to different psychological functions , that occur in relation to the external context  and in relation to the internal context . Therefore, in some cases this behaviour it can be functionally adaptive to the context or in other cases it can be highly dysfunctional to the context and have negative consequence on one’s own and others’ health. The ability to adapt is closely linked to the degree of psychological flexibility, defined as acting in accordance with personal goals and values, in the presence of potentially interfering thoughts and feelings, and with a greater appreciation of what their current situation or context allows .Moreover, recent studies have shown that psychological flexibility mitigated the detrimental impacts of the pandemic on mental health, peritraumatic distress, anxiety, depression, insomnia and facets of psychological inflexibility exacerbated the impact of these risks ,20,21.As such, the aim of this study is to investigate the psychosocial factors contributing to the cancellation of medical appointments, by patients with pre-existing health conditions, in Italy, during the SARS-CoV-2 outbreak.We have developed a cross-sectional study in which anonymous questionnaires were implemented to assess the impact of the SARS-CoV-2 outbreak on compliance with medical treatment and on adherence to a healthy lifestyle, amongst patients with pre-existing medical conditions. The questionnaires could be completed as a hard copy or as an electronic version, online. Hospital inpatients and outpatients were the first potential participants to be made aware of the study. A “snowball sampling strategy”, focusing on recruiting other participants with pre-existing medical conditions of mainland Italy, then followed. The study involved eleven Italian hospitals located throughout Italy, including northern, central and southern regions.Data were collected during the last week of lockdown (20–27 April 2020) (T1 phase), and again one month later (22–30 May 2020) (T2 phase), from individuals aged eighteen years or over with pre-existing medical conditions, and who were not afflicted with COVID-19 at the time of data collection. Potential respondents were made aware of the survey by other study participants, or by reading information about the study that was made available to them during a hospital visit. Potential respondents were invited to complete questionnaires online or as hard copies for patients visiting the hospital. All participants provided informed consent to participate in the anonymous survey. The procedures were clearly explained in writing, and participants could withdraw from the study at any time without explaining their reasons for doing so. Expedited ethics approval was obtained from the Institutional Review Board of participants, the Ethical Committee for Clinical Trials of the Provinces of Verona and Rovigo in Northern Italy (Prog. 2642CESC), and the Local Ethics Committees of Centres that collaborated in this study, which conformed to the principles embodied in the Declaration of Helsinki. Information about this study was posted on a dedicated website of the Clinical Psychological Service of the Scientific Institute for Research Hospitalisation and Health Care (IRCSS) Sacro Cuore Don Calabria Hospital in Negrar.The following sociodemographic and contextual data were collected: gender, age, level of education, residential location, marital status, employment status, economic losses relating to the SARS-CoV-2 outbreak, parental status, family composition, cohabitation status before and during lockdown, compliance with physical isolation regulations, and the type of medical pathology suffered. Respondents were also asked about their sources of information regarding SARS-CoV-2 infection rates as well as COVID-19 symptom management. They were also asked to rate their trust in these sources, and about their main sources of psychological support. Information regarding lifestyle, interpersonal relationships and psychological trauma relating to the SARS-CoV-2 outbreak, . Data were also collected regarding physical health status and treatment phase . Data about access to health services during lockdown specifically regarding the location of medical centres, clinic-based consultations, admission to hospital, and distress relating to isolation during lockdown were also collected. Data were also collected regarding mental health status psychiatric history, the emotional impact of the SARS-CoV-2 outbreak, any behaviours stemming from a fear of contracting SARS-CoV-2, and any other lifestyle choices that may impact one’s general health, for example, alcohol consumption or smoking status.In order to evaluate the behaviour of patients in a stressful situation, in this instance, the SARS-CoV-2 outbreak, we developed the “ImpACT” 31-item self-report questionnaire. The theoretical model of reference used was the functional contextualism and the Relational Frame Theory (RFT)  that encIn general terms cognitive fusion refers to excessive or improper regulation of behaviour by verbal processes, such as rules and derived relational networks. When cognitive fusion increases, human behaviour is less sensitive to environmental contingencies. As a result, people may act in a way that is inconsistent with what the environment affords relevant to chosen values and goals .Experiential avoidance is the attempt to alter the form, frequency, or situational sensitivity of private events even when doing so causes behavioural harm . Due to Hayes et al.  propose In the ImpACT questionnaire, the word “COVID-19” was used instead of “stressful situation”, in order to identify specific contextual behavioural responses .The traumatic and stressful psychological impact of COVID-19 was measured using the Impact of Event Scale-Revised (IES-R) . The IESDepression, anxiety and stress, defined as irritability, nervous tension, difficulty relaxing, and agitation, were measured using the Depression, Anxiety and Stress Scale (DASS-21) self-report questionnaire . The totCognitive fusion, described as the degree to which an individual becomes caught up in their thoughts, was measured using the Cognitive Fusion Questionnaire (CFQ) . Learninp-value < 0.2) to cancelling an appointment with a specialist were included in the full logistic regression model. Model-building strategies included checking for convergence, correlation and goodness-of-fit test. The likelihood-ratio test was used to compare candidate models. A statistical significance level of 0.05 was adopted for all tests.All analyses were performed on patients with pre-existing medical conditions using R software, version 3.6.1 . All 31 Among 884 respondents in the first round of questionnaires, 126 were excluded because did not fulfil the inclusion criteria . Out of 817 respondents during the second round of questionnaires, 119 were excluded because of presence of exclusion criteria . Therefore, the final sample consisted of 758 patients with pre-existing medical conditions in T1 and 698 in T2.The sociodemographic characteristics of these sample from the lockdown phase (T1) and the post lockdown phase (T2) are shown in Some of the reported pre-existing medical conditions were: cancer (27.0% in T1 and 33.2% in T2), rheumatic disease (15.8% in T1 and 8.2% in T2), multiple sclerosis (13.7% in T1 and 10.4% in T2), cardiovascular disease (9.9% in T1 and 6.3% in T2) and HIV (3.8% in T1 and 11.5% in T2). Finally, 72.0% subjects reported the official websites of national institutions as being the most reliable source of information regarding SARS-CoV-2 and COVID-19 during the first round of questionnaire and 63.8% in the second. This was followed by doctors, nurses or psychologists (44.3% in T1 and 46.4% in T2), and online newspapers (25.9% in T1 and 24.6% in T2). Respondents considered social media (19.0% in T1 and 13.3% in T2) and instant messaging apps (7.5% in T1 and 5.9% in T2) to be trusted sources of information as well.Data regarding the psychological impact of the SARS-CoV-2 outbreak on individuals with pre-existing medical conditions are shown in Further to this, 35.2% individuals stated to have cancelled an appointment with their specialist doctor due to fear of COVID-19 in the lockdown phase and 29.4% in the post lockdown phase.Results from the multivariate analysis show that during the lockdown (T1), married  and widowed subjects  were more likely to cancel a medical appointment than single subjects. However, age, gender, level of education and employment status were not associated with compliance to medical appointments. Further to this, after lockdown (T2), no differences were found between the various sociodemographic subgroups regarding compliance with medical appointments.Analysis of the lockdown (T1) phase revealed a significant association between post-traumatic stress symptoms (IES-R scores) and the likelihood of cancelling a medical appointment . This association was not apparent in the second, post-lockdown (T2) phase.Other psychological variables, including depression, anxiety, and stress (DASS-21), were not associated cancelling medical appointments . The likelihood of a patient cancelling a medical appointment if they had spoken with a specialist over the phone during the lockdown (T1) was 2.24 times higher than if they had not spoken to a specialist (95% CI = 1.28–3.95). Similar results were found for subjects interviewed after the lockdown (T2) whereby patients that had spoken to a specialist were 2.13 times more likely to cancel a medical appointment (95% CI = 1.16–3.97) .In addition, the concern that medical staff would develop COVID-19 and be too unwell to be able to treat subjects effectively, was significantly associated with cancelling appointments in both phases. Patients who were worried about encountering contagious medical staff, were 2.17 times more likely to cancel a medical appointment during T1 phase (95% CI = 1.25–3.82) and 3.41 times more likely to do so during T2 phase (95% CI = 1.83–6.51) than patients that did not have this concern.During the lockdown phase (T1), patients who felt more alone than usual showed a significantly higher likelihood of cancelling a medical appointment as opposed to patients who did not feel alone . Further to this, subjects who kept in contact with fellow patients were 2.13 times more likely to cancel their medical appointment than those who were not in contact with other patients (95% CI = 1.25–3.64) .A significant association was observed amongst patients regarding increased food intake, increased alcohol consumption and/or increased numbers of cigarettes smoked, as coping strategies to manage emotions such as, boredom, apathy, and/or sadness. The likelihood of a subject that uses such strategies would cancel a medical appointment was 1.73 times higher than for subjects that did not adopt such strategies during lockdown (T1) (95% CI = 1.03–2.90) and 2.57 times higher post lockdown (T2) (95% CI = 1.43–4.68).Our results indicate that the SARS-CoV-2 outbreak, and resultant lockdown, have had a significant psychological impact on individuals with pre-existing medical conditions. About a third of the sample reported severe post-traumatic symptoms, 19.4% severe to extremely severe stress symptoms, about 15% reported severe to extremely severe anxiety, and 13% severe to extremely severe depression (during lockdown (T1) and similar data after (T2)).Psychological distress appears to be linked to a fear of contracting SARS-CoV-2 and developing COVID-19, which has affected medical appointment attendance and disrupted disease management approaches. Fear of COVID-19 was apparent in many subjects in both phases of the study (during lockdown and after) and was found to correlate with high post-traumatic stress scores. Of note, subjects with HIV did not show any greater propensity to cancel medical appointments due to concerns about COVID-19 as compared to subjects with cancer, rheumatic disease, multiple sclerosis, chronic migraine, cardiovascular disease, endocrine diseases, gynaecological disorders, or others .Failure to attend medical appointments is a problem commonly encountered by clinicians in an ambulatory setting. Other than the obvious disruption to working practices, and waste of resources, a patient’s disease management can also be adversely affected by such behaviour . From ouIn summary, during the lockdown, some factors accounted for an increased likelihood in cancelling a medical appointment: the psychological impact of the SARS-CoV-2 outbreak as measured by IES-R, marital status , feelings of loneliness, and a set of different behavioural patterns of patients or their doctors, identified by the ImpACT questionnaire. It should be noted that for this study the Impact questionnaire was not used to evaluate psychological flexibility but was used considering every single item. Specifically, our results have shown that the behaviour of cancelling medical appointments was more apparent amongst subjects who contacted their medical specialist more often; in addition, this behaviour of cancelling medical appointments may have been further influenced by the community to which a patient belongs and reinforced particular by remaining in contact with other patients. Furthermore, patients who were afraid of medical staff becoming unwell and being unable to provide adequate treatment, were also more likely to cancel a medical appointment.Some pattern of behaviours may reflect a dysfunctional use of experiential avoidance, as for i.e., the behaviour of asking reassurance may reflect an attempt to avoid or control the fear , or the behaviour of search more often food, drink to avoid negative emotions ). Patients who cancelled medical appointments were more likely to engage in such behaviours both during and after lockdown. Emotional problems and dysfunctional behaviour often manifest together and interact reciprocally over time, for example, depression often predicts the onset of eating disorders, and eating disorders can induce depression ,37,38. SOverall, our results suggest that the decision to cancel a medical appointment is multifactorial, including psychological, sociodemographic status, relationship to health professional and isolation, and can be linked to several variables that affect patient behaviour in various ways.Although we cannot clearly define what consequences cancelling medical appointments may have on disease progression, our results do suggest that such behaviour are related to an unhealthy and dysfunctional use of experiential avoidance and is a result of a fear of COVID-19.According to ACT, psychological flexibility may be considered the key construct of psychological well-being and resiliency. Psychological flexibility enables an individual to alter their behaviour when it compromises their personal values, or is required to adapt to changing circumstances . It invoOur findings raise several questions about the delivery of care to patients who are afraid to attend a medical appointment. Fear may explain the higher rate of failing to attend follow-up appointments, particularly in cases where the patient has serious concerns about their illness and their continuity of care, or if the patient experiences high levels of distress or psychological issues, such as, anxiety, depression, or PTSD.Furthermore, the results obtained from this study could be useful for implementing future guidelines or recommendations addressed to patients  and addrThe results of our research highlight that patients who tend to cancel medical appointments are also those who suffer from serious psychological problems, such as, PTSD or suicidal thoughts. Such individuals adopt avoidance behaviours, and unhealthy dysfunctional behaviours.Post-traumatic stress symptoms have led to behaviours that attempt to control the aversive and fear-inducing situation , and other behaviours aimed at avoiding the aversive situation .In situations of emergency, such as the SARS-CoV-2 outbreak, it may be useful to invest more in the psychological management of patients with pre-existing diseases, who may develop a heightened state of alertness and anxiety that could develop into PTSD. It is extremely important to identify the most vulnerable patients early on in order to provide adequate and effective support, this can be achieved by phone contact with especially anxious patients that refuse to attend appointments. It is also essential to support patients already diagnosed with PTSD, as the likelihood of clinical worsening in such scenarios as that of the SARS-CoV-2 outbreak is high. This would have direct repercussions on the patient’s quality of life, adherence to therapy, and on the progression of their disease.During lockdown, many patients did not have sufficient access to psychotherapy and were left to manage their feelings without clinical support. As a result of this, pervasive dysfunctional behavioural patterns, such as, avoidance behaviours and reduced social contact were adopted, which increased the likelihood of cancelling medical appointments during the lockdown and after.Understanding the underlying causes of cancelled medical appointments will allow time and valuable resources to be better utilised so that all patients may receive a higher standard of care.Our findings offer an important contribution to our understanding of the psychological reaction of patients with pre-existing medical conditions during lockdowns and subsequent phases of a pandemic by describing the type and extent of distress experienced by patients and the impact of this on compliance with disease management requirements. Further study should aim to develop targeted and timely intervention to ensure the continued monitoring of especially vulnerable patients afflicted with medical disease.The main strength of this study is our focus on patients with pre-existing medical conditions. As such, the majority of subjects were patients known to hospital services; this is a particularly relevant to online questionnaires, in which the true identity of respondents can be difficult to ascertain. However, the “snowball sampling strategy” as a recruitment method may be a limitation of the study due to possible selection bias for more compliant patients that are probably less likely to cancel medical appointments. A further limitation of the study may be linked to the external context , that may have influenced the cancellation of appointments, an aspect that could be further evaluated in future studies. However, alternative methodologies were limited given the circumstances of an unexpected and sudden pandemic. A further limitation is the lack of any baseline evaluation of subjects before the pandemic, but this limitation is partially overcome by the comparison of the mean scores found in previous studies ,23.