A Novel SPR Immunosensor Based on Dual Signal Amplification Strategy for Detection of SARS-CoV-2 Nucleocapsid Protein.

Since the global outbreak of coronavirus disease 2019 (COVID-19), it has spread rapidly around the world. The nucleocapsid (N) protein is one of the most abundant SARS-CoV-2 proteins. Therefore, a sensitive and effective detection method for SARS-CoV-2 N protein is the focus of research. Here, we developed a surface plasmon resonance (SPR) biosensor based on the dual signal-amplification strategy of Au@Ag@Au nanoparticles (NPs) and graphene oxide (GO). Additionally, a sandwich immunoassay was utilized to sensitively and efficiently detect SARS-CoV-2 N protein. On the one hand, Au@Ag@Au NPs have a high refractive index and the capability to electromagnetically couple with the plasma waves propagating on the surface of gold film, which are harnessed for amplifying the SPR response signal. On the other hand, GO, which has the large specific surface area and the abundant oxygen-containing functional groups, could provide unique light absorption bands that can enhance plasmonic coupling to further amplify the SPR response signal. The proposed biosensor could efficiently detect SARS-CoV-2 N protein for 15 min and the detection limit for SARS-CoV-2 N protein was 0.083 ng/mL, with a linear range of 0.1 ng/mL~1000 ng/mL. This novel method can meet the analytical requirements of artificial saliva simulated samples, and the developed biosensor had a good anti-interference capability.

Case report: Acute hepatitis in neonates with COVID-19 during the Omicron SARS-CoV-2 variant wave: a report of four cases.

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), first emerging in December 2019 and continuously evolving, poses a considerable challenge worldwide. It was reported in the literature that neonates had mild upper respiratory symptoms and a better outcome after Omicron SARS-CoV-2 variant infection, but there was insufficient data about complications and prognosis. Case Presentation: In this paper, we present the clinical and laboratory characteristics of four COVID-19 neonate patients with acute hepatitis during the Omicron SARS-CoV-2 variant wave. All patients had a clear history of Omicron exposure and were infected via contact with confirmed caregivers. Low to moderate fever and respiratory symptoms were the primary clinical manifestations, and all patients had a normal liver function at the initial stage of the course. Then, the fever lasted 2 to 4 days, and it was noted that hepatic dysfunction might have occurred 5 to 8 days after the first onset of fever, mainly characterized by moderate ALT and AST elevation (>3 to 10-fold of upper limit). There were no abnormalities in bilirubin levels, blood ammonia, protein synthesis, lipid metabolism, and coagulation. All the patients received hepatoprotective therapy, and transaminase levels gradually decreased to the normal range after 2 to 3 weeks without other complications. Conclusions: This is the first case series about moderate to severe hepatitis in COVID-19 neonatal patients via horizontal transmission. Besides fever and respiratory symptoms, the clinical doctor should pay much attention to evaluating the risk of liver function injury after SARS-CoV-2 variants infection, which is usually asymptomatic and has a delayed onset.

Wastewater surveillance and an automated robot: effectively tracking SARS-CoV-2 transmission in the post-epidemic era.

Wastewater-based epidemiology (WBE) has exhibited great utility in the early and rapid identification of SARS-CoV-2. However, the efficacy of wastewater surveillance under China's previous strict epidemic prevention policy remains to be described. We collected the WBE data of wastewater treatment plants (WWTPs) in the Third People's Hospital of Shenzhen and several communities to determine the significant effectiveness of routine wastewater surveillance in monitoring the local spread of SARS-CoV-2 under tight containment of the epidemic. The results of 1 month of continuous wastewater surveillance showed that positive signals for SARS-CoV-2 RNA were detected in the wastewater samples, and a significant positive correlation was observed between the virus concentration and the number of daily cases. In addition, the community's domestic wastewater surveillance results were confirmed even 3 days before, or simultaneously with, the infected patient being confirmed as having the virus. Meanwhile, an automated sewage virus detection robot, ShenNong No.1 robot, was developed, showing a high degree of agreement with experimental data, offering the possibility of large-scale multi-point surveillance. Overall, our results illustrated the clear indicative role of wastewater surveillance in combating COVID-19 and provided a practical basis for rapidly expanding the feasibility and value of routine wastewater surveillance for future emerging infectious diseases.

Clinical risk score for early prediction of recurring SARS-CoV-2 positivity in non-critical patients.

Introduction: Recurrent positive results in quantitative reverse transcriptase-PCR (qRT-PCR) tests have been commonly observed in COVID-19 patients. We aimed to construct and validate a reliable risk stratification tool for early predictions of non-critical COVID-19 survivors' risk of getting tested re-positive within 30 days. Methods: We enrolled and retrospectively analyzed the demographic data and clinical characters of 23,145 laboratory-confirmed cases with non-critical COVID-19. Participants were followed for 30 days and randomly allocated to either a training (60%) or a validation (40%) cohort. Multivariate logistic regression models were employed to identify possible risk factors with the SARS-CoV-2 recurrent positivity and then incorporated into the nomogram. Results: The study showed that the overall proportion of re-positive cases within 30 days of the last negative test was 24.1%. In the training cohort, significantly contributing variables associated with the 30-day re-positivity were clinical type, COVID-19 vaccination status, myalgia, headache, admission time, and first negative conversion, which were integrated to build a nomogram and subsequently translate these scores into an online publicly available risk calculator (https://anananan1.shinyapps.io/DynNomapp2/). The AUC in the training cohort was 0.719 [95% confidence interval (CI), 0.712-0.727] with a sensitivity of 66.52% (95% CI, 65.73-67.30) and a specificity of 67.74% (95% CI, 66.97-68.52). A significant AUC of 0.716 (95% CI, 0.706-0.725) was obtained for the validation cohort with a sensitivity of 62.29% (95% CI, 61.30-63.28) and a specificity of 71.26% (95% CI, 70.34-72.18). The calibration curve exhibited a good coherence between the actual observation and predicted outcomes. Conclusion: The risk model can help identify and take proper management in high-risk individuals toward the containment of the pandemic in the community.

Mental Health Among University Students, Using the 12-item General Health Questionnaire.

Context: To date, researchers have found that poor mental health was common during the COVID-19 epidemic. Even if they had been relatively resistant to suicidal ideation during the first three waves of the pandemic, university students may experience a delayed impact on their mental health. Objective: The study intended to measure mental health among university students in Wuhu City, China and to identify an effective approach to universities can use to prevent mental-health issues. Design: The research team performed a cross-sectional study. Setting: The study took place at Anhui polytechnic university, Wuhu, China. Participants: Participants were 2371 students at Anhui polytechnic university in Wuhu city, China. Outcome Measures: The research team used the two-item General Health Questionnaire-12 (GHQ-12) to measure participants' mental health. Results: Among the 2371 participants, 1727 had poor mental health (72.84%), including 843 males (48.81%) and 884 females (51.19%). Poor mental health was significantly associated with an urban residential location (P > .01), the female gender (p>0.01), the second school year (P > .01), and the parents' education level of junior high school or below (both P > .01). Conclusions: The current study suggests that poor mental health among university students is common. Being female, from an urban area, and in the second year of school and having parents with an education of junior high school or below had poorer mental health than those who were male, from the countryside, and in the first year of school and who had parents with a higher level of education.

Little to no expression of angiotensin-converting enzyme-2 on most human peripheral blood immune cells but highly expressed on tissue macrophages.

Angiotensin-converting enzyme-2 (ACE2) has been recognized as the binding receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Flow cytometry demonstrated that there was little to no expression of ACE2 on most of the human peripheral blood-derived immune cells including CD4+ T, CD8+ T, activated CD4+ /CD8+ T, Tregs, Th17, NKT, B, NK cells, monocytes, dendritic cells, and granulocytes. There was no ACE2 expression on platelets and very low level of ACE2 protein expression on the surface of human primary pulmonary alveolar epithelial cells. The ACE2 expression was markedly upregulated on the activated type 1 macrophages (M1). Immunohistochemistry demonstrated high expressions of ACE2 on human tissue macrophages, such as alveolar macrophages, Kupffer cells within livers, and microglial cells in brain at steady state. The data suggest that alveolar macrophages, as the frontline immune cells, may be directly targeted by the SARS-CoV-2 infection and therefore need to be considered for the prevention and treatment of COVID-19.

DIA-based technology explores hub pathways and biomarkers of neurological recovery in ischemic stroke after rehabilitation.

Objective: Ischemic stroke (IS) is a common disease that causes severe and long-term neurological disability in people worldwide. Although rehabilitation is indispensable to promote neurological recovery in ischemic stroke, it is limited to providing a timely and efficient reference for developing and adjusting treatment strategies because neurological assessment after stroke treatment is mostly performed using scales and imaging. Therefore, there is an urgent need to find biomarkers that can help us evaluate and optimize the treatment plan. Methods: We used data-independent acquisition (DIA) technology to screen differentially expressed proteins (DEPs) before and after ischemic stroke rehabilitation treatment, and then performed Gene Ontology (GO) and pathway enrichment analysis of DEPs using bioinformatics tools such as KEGG pathway and Reactome. In addition, the protein-protein interaction (PPI) network and modularity analysis of DEPs were integrated to identify the hub proteins (genes) and hub signaling pathways for neurological recovery in ischemic stroke. PRM-targeted proteomics was also used to validate some of the screened proteins of interest. Results: Analyzing the serum protein expression profiles before and after rehabilitation, we identified 22 DEPs that were upregulated and downregulated each. Through GO and pathway enrichment analysis and subsequent PPI network analysis constructed using STRING data and subsequent Cytoscape MCODE analysis, we identified that complement-related pathways, lipoprotein-related functions and effects, thrombosis and hemostasis, coronavirus disease (COVID-19), and inflammatory and immune pathways are the major pathways involved in the improvement of neurological function after stroke rehabilitation. Conclusion: Complement-related pathways, lipoprotein-related functions and effects, thrombosis and hemostasis, coronavirus disease (COVID-19), and inflammation and immunity pathways are not only key pathways in the pathogenesis of ischemic stroke but also the main pathways of action of rehabilitation therapy. In addition, IGHA1, LRG1, IGHV3-64D, and CP are upregulated in patients with ischemic stroke and downregulated after rehabilitation, which may be used as biomarkers to monitor neurological impairment and recovery after stroke.

A fast RT-qPCR system significantly shortens the time for SARS-CoV-2 nucleic acid test.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to global development. Rapid and accurate diagnosis is critical for containing the pandemic and treating patients in time. As the gold standard for SARS-CoV-2 diagnosis, the qualitative reverse transcription-PCR (RT-qPCR) test has long been criticized for its long detection time. In this study, we optimized the primers and probes targeting SARS-CoV-2 ORF1ab and N gene designed by the Chinese Center for Disease Control and Preventions (CDC) to increase their Tm values to meet the optimal elongation temperature of Taq DNA polymerase, thus greatly shortened the elongation time. The higher elongation temperature in turn narrowed the temperature range of the reaction and saved more time. In addition, by shortening the distance between the fluorophore at the 5' end and the quencher in the middle we got a probe with higher signal-to-noise ratio. Finally, by using all these measures and optimized RT-qPCR program we successfully reduced the time (nucleic acid extraction step is not included) for nucleic acid test from 74 min to 26 min.

Small change for big improvement in the preparation of the key intermediate N 1, N 3-disubstituted 1,3,5-triazone of ensitrelvir.

In this study, the key intermediate N 1, N 3-disubstituted 1,3,5-triazone of ensitrelvir fumaric acid, approved in Japan for the treatment of SARS-CoV-2 infection under the emergency regulatory approval system, was produced from S-ethylisothiourea hydrobromide and aminomethyl triazole with CDI by four-step telescoped strategy including CDI-activated, condensation, CDI-cyclization, and N 1-alkylation. The strategy with simple conditions and operations had a total yield of 53% on a gram scale. The strategy for synthesizing the key N 1, N 3-disubstituted 1,3,5-triazone intermediate of ensitrelvir might provide a new avenue for further research and development of ensitrelvir analogs.

A photoelectrochemical immunosensor based on magnetic all-solid-state Z-scheme heterojunction for SARS-CoV-2 nucleocapsid protein detection.

Rapid, convenient and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is urgently needed to timely diagnosis of coronavirus pandemic (COVID-19) and control of the epidemic. In this study, a signal-off photoelectrochemical (PEC) immunosensor was constructed for SARS-CoV-2 nucleocapsid (N) protein detection based on a magnetic all-solid-state Z-scheme heterojunction (Fe3O4@SiO2@TiO2@CdS/Au, FSTCA). Integrating the advantages of magnetic materials and all-solid-state Z-scheme heterostructures, FSTCA was implemented to ligate the capture antibody to form magnetic capture probe (FSTCA/Ab1). It can simplify the separation and washing process to improve reproducibility and stability, while allowing immune recognition to be performed in the liquid phase instead of the traditional solid-liquid interface to improve anti-interference. Besides, the heterojunction inhibited the recombination of photogenerated electron/hole (e-/h+) and promoted the light absorption to provide superior photoelectric substrate signal. The mechanism of photogenerated e-/h+ transfer of FSTCA were investigated by the electron spin resonance (ESR) spectroscopy. SiO2 spheres loaded with Au NPs utilized as an efficient signal quencher. The steric hindrance effect of SiO2@Au labeled detection antibodies (SiO2@Au-Ab2) conjugates significantly diminished light absorption and hindered the transfer of photogenerated electrons, further amplifying the signal change value. Based on the above merits, the elaborated immunosensor had a wide linear range of 10 pg mL-1-100 ng mL-1 and a low detection limit down to 2.9 pg mL-1 (S/N = 3). The fabricated PEC immunosensor demonstrated strong anti-interference, easy operation, and high sensitivity, showing enormous potential in clinical diagnosis of SARS-CoV-2.

Sandwich mode lateral flow assay for point-of-care detecting SARS-CoV-2

The global corona virus disease 2019 (COVID-19) has been announced a pandemic outbreak, and has threatened human life and health seriously. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as its causative pathogen, is widely detected in the screening of COVID-19 patients, infected people and contaminated substances. Lateral flow assay (LFA) is a popular point-of-care detection method, possesses advantages of quick response, simple operation mode, portable device, and low cost. Based on the above advantages, LFA has been widely developed for detecting SARS-CoV-2. In this review, we summarized the articles about the sandwich mode LFA detecting SARS-CoV-2, classified according to the target detection objects indicating genes, nucleocapsid protein, spike protein, and specific antibodies of SARS-CoV-2. In each part, LFA is further classified and summarized according to different signal detection types. Additionally, the properties of the targets were introduced to clarify their detection significance. The review is expected to provide a helpful guide for LFA sensitization and marker selection of SARS-CoV-2. [Display omitted] • LFA as a simple user friendly tool is widely employed for SARS-CoV-2 detection. • Present review focus on latest developments in LFAs to detect SARS-CoV-2. • Sensitization strategies and marker properties are beneficial to further research. [ FROM AUTHOR]

Small change for big improvement in the preparation of the key intermediate N1, N3-disubstituted 1,3,5-triazone of ensitrelvir† † Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2ra07844a

In this study, the key intermediate N1, N3-disubstituted 1,3,5-triazone of ensitrelvir fumaric acid, approved in Japan for the treatment of SARS-CoV-2 infection under the emergency regulatory approval system, was produced from S-ethylisothiourea hydrobromide and aminomethyl triazole with CDI by four-step telescoped strategy including CDI-activated, condensation, CDI-cyclization, and N1-alkylation. The strategy with simple conditions and operations had a total yield of 53% on a gram scale. The strategy for synthesizing the key N1, N3-disubstituted 1,3,5-triazone intermediate of ensitrelvir might provide a new avenue for further research and development of ensitrelvir analogs. A four-step telescoped strategy for synthesis of the key intermediate of ensitrelvir, approved in Japan for the treatment of SARS-CoV-2 infection under the emergency regulatory approval system, was developed.

Safety and immunogenicity of COVID-19 vaccination in immunocompromised patients.

ABSTRACT: The coronavirus disease 2019 (COVID-19) pandemic poses a great threat to public health. Individuals who are immunocompromised because of the progression of the primary disease or receiving immunosuppressive medications are prone to severe COVID-19 complications and poor outcomes. Abundant data have shown that many COVID-19 vaccines are safe and effective in large-scale populations; however, these clinical trials have excluded immunocompromised populations. Available evidence indicates that immunocompromised populations have a blunted immune response to other vaccines, raising concerns regarding the efficacy of COVID-19 vaccination in these populations. Thus, there is an urgent need to delineate the efficacy of COVID-19 vaccines in these vulnerable populations. Here, we review the characteristics of specific humoral and cellular responses to COVID-19 vaccination in immunocompromised populations, including HIV-infected patients and those receiving immunosuppressive treatment, especially solid organ transplant recipients and those undergoing anti-CD20 treatment. We also addressed the challenges that immunocompromised populations will face in the future pandemic and the need for basic and clinical translational studies to highlight the best vaccination strategies for these populations.

Mental Health Among University Students, Using the 12-item General Health Questionnaire.

Context: To date, researchers have found that poor mental health was common during the COVID-19 epidemic. Even if they had been relatively resistant to suicidal ideation during the first three waves of the pandemic, university students may experience a delayed impact on their mental health. Objective: The study intended to measure mental health among university students in Wuhu City, China and to identify an effective approach to universities can use to prevent mental-health issues. Design: The research team performed a cross-sectional study. Setting: The study took place at Anhui polytechnic university, Wuhu, China. Participants: Participants were 2371 students at Anhui polytechnic university in Wuhu city, China. Outcome Measures: The research team used the two-item General Health Questionnaire-12 (GHQ-12) to measure participants' mental health. Results: Among the 2371 participants, 1727 had poor mental health (72.84%), including 843 males (48.81%) and 884 females (51.19%). Poor mental health was significantly associated with an urban residential location (P > .01), the female gender (p>0.01), the second school year (P > .01), and the parents' education level of junior high school or below (both P > .01). Conclusions: The current study suggests that poor mental health among university students is common. Being female, from an urban area, and in the second year of school and having parents with an education of junior high school or below had poorer mental health than those who were male, from the countryside, and in the first year of school and who had parents with a higher level of education.

Two-stage one-pot synthetic strategy for the key triazone-triazole intermediate of ensitrelvir (S-217622), an oral clinical candidate for treating COVID-19.

Herein, the preparation of the key triazone-triazole intermediate of ensitrelvir (S-217622) via sequential cyclization and alkylation reaction is described. Firstly, chloromethyl triazole was synthesized through a one-pot tandem process (condensation and cyclization reaction) from commercially available chloroacetamide in a 72% yield. Then, the key triazone-triazole intermediate was obtained in a second one-pot process by N-alkylation with triazone followed by highly selective N 1-methylation with iodomethane in a 54% yield. In addition, two of the main process impurities were synthesized and identified. This novel alternative two-stage one-pot strategy for synthesizing the key triazone-triazole intermediate opens a new avenue for further research and development of ensitrelvir analogs.

CdTe QDs-sensitized TiO2 nanocomposite for magnetic-assisted photoelectrochemical immunoassay of SARS-CoV-2 nucleocapsid protein.

A sensitive, reliable, and cost-effective detection for SARS-CoV-2 was urgently needed due to the rapid spread of COVID-19. Here, a "signal-on" magnetic-assisted PEC immunosensor was constructed for the quantitative detection of SARS-CoV-2 nucleocapsid (N) protein based on Z-scheme heterojunction. Fe3O4@SiO2@Au was used to connect the capture antibody to act as a capture probe (Fe3O4@SiO2@Au/Ab1). It can extract target analytes selectively in complex samples and multiple electrode rinsing and assembly steps were avoided effectively. CdTe QDs sensitized TiO2 coated on the surface of SiO2 spheres to form Z-scheme heterojunction (SiO2@TiO2@CdTe QDs), which broadened the optical absorption range and inhibited the quick recombination of photogenerated electron/hole of the composite. With fascinating photoelectric conversion performance, SiO2@TiO2@CdTe QDs were utilized as a signal label, thus further realizing signal amplification. The migration mechanism of photogenerated electrons was further deduced by active material quenching experiment and electron spin resonance (ESR) measurement. The elaborated immunosensor can detect SARS-CoV-2 N protein in the linear range of 0.005-50 ng mL-1 with a low detection limit of 1.8 pg mL-1 (S/N = 3). The immunosensor displays extraordinary sensitivity, strong anti-interference, and high reproducibility in detecting SARS-CoV-2 N protein, which envisages its potential application in the clinical diagnosis of COVID-19.

Trimetallic Au@Pd@Pt nanozyme-enhanced lateral flow immunoassay for the detection of SARS-CoV-2 nucleocapsid protein.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seriously threatened global public health. Establishing a rapid and sensitive diagnostic test for early detection of the SARS-CoV-2 nucleocapsid protein is urgently required to defend against the pandemic. Herein, an enhanced lateral flow immunoassay (LFIA) was fabricated by trimetallic Au@Pd@Pt core-shell nanozymes for detection of the SARS-CoV-2 nucleocapsid protein. The Au@Pd@Pt nanozymes (Au@Pd@Pt NZs) synthesized via a one-pot method, with a dendrite morphology and uniform particle size, showed excellent peroxidase-like activity. Due to the perfect enzyme-like catalytic activity toward 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2), the catalytic signal could be generated even by a tiny amount of Au@Pd@Pt NZs accumulated on the test strip. Therefore, rapid detection with higher sensitivity was achieved. The Au@Pd@Pt NZs-based LFIA provided a quantitative range of 0.05-100 ng mL-1 with a limit of detection of 0.037 ng mL-1, which was 17-fold lower than the LFIA without enhancement. The average recoveries from spiked samples were in the range of 92.5-107.9% with relative standard deviations all less than 4%, indicating the reliability and repeatability of the proposed LFIA. Additionally, the proposed LFIA could report results within 30 min using a microplate reader. In conclusion, the Au@Pd@Pt NZs-LFIA is a rapid, simple, and sensitive method for detecting the SARS-CoV-2 nucleocapsid protein.

Determinants of the willingness of medical staff to vaccinate their children with a booster dose of the COVID-19 vaccine in Taizhou, China.

The study aimed to determine the willingness of medical staff to have their children vaccinated with a COVID-19 booster in Taizhou, China. From March 21 to April 19, 2022, an online questionnaire survey was conducted to assess the willingness of medical staff to vaccinate their children with a booster dose of the COVID-19 vaccine. Of the 1,252 medical staff in a tertiary grade hospital in Taizhou who were invited to answer the structured questionnaire, 514 (41.1%) samples had valid information for further data analysis. Four hundred thirty-seven medical staff (85.0%) were willing to have their children receive vaccine boosters. After adjustments for confounding factors, the opinion ('Do you think your child needs a booster vaccination against COVID-19?') (yes vs. no, OR = 6.91, 95% CI: 3.29-14.54), the viewpoint ('What are your thoughts the effectiveness of COVID-19 vaccine boosters for children?' (≥12 vs. <12, OR = 13.81, 95% CI: 4.03-), and the attitude ('Your attitude to whether your child is boosting the Covid-19 vaccine?') (yes vs. no, OR = 4.66, 95% CI: 2.30-9.44) were significantly associated with their willingness to have their children receive a COVID-19 vaccine booster. A moderate percentage of the respondents expressed willingness to have their children receive booster vaccines. The findings implied that factors affecting medical staffs' willingness to vaccinate their children with a COVID-19 vaccine booster included viewpoint, opinion, and attitudes.

Photoacoustic detection of SARS-CoV-2 spike N501Y single-nucleotide polymorphism based on branched rolling circle amplification

Rapid and accurate diagnosis of SARS-CoV-2 single-nucleotide variations is an urgent need for the initial detection of local circulation and monitoring the alternation of dominant variant. In this proof-of-concept study, a homogeneous and isothermal photoacoustic biosensor is demonstrated for rapid molecular amplification and detection of a synthetic DNA corresponding to SARS-CoV-2 spike N501Y. Branched rolling circle amplification produces single-stranded amplicons that can aggregate detection probe-modified AuNPs, which induces a strong photoacoustic signal at 640 nm due to both the surface plasmon resonance shift and the size-dependent effect of laser-induced nanobubbles, achieving a sub-femtomolar detection limit within a total assay time of 80 min. The limit of detection can be kept when measuring 5% serum samples. Moreover, the proposed biosensor is highly specific for single-nucleotide polymorphism discrimination and robust against background DNA.