Rapid diagnostics: the detection of neuraminidase activity as a technology for high-specificity targets.

The accurate detection of influenza by clinical symptoms is challenging since multiple pathogenic viruses and bacteria mimic similar symptoms in a patient. With new and more effective influenza therapeutics available, there is a growing need for highly accurate and rapid diagnosis of influenza, particularly when the window of opportunity for proper treatment is measured in hours. A parallel technology, which is also used in the treatment of influenza, was developed for the rapid diagnosis of influenza by exploiting the enzymatic activity of influenza neuraminidase. This technology, which is called Pathozyme, offers the high specificity inherent from the conservation of the neuraminidase active site. The ZstatFlu test uses a small molecule derivative of sialic acid chemically coupled to a reporter group together with simple point-of-care reagents for directly detecting influenza from a patient specimen with high specificity. A second-generation platform technology using this neuraminidase detection system coupled with a more sensitive chemiluminescent reporter has been developed and formatted for reading on high-speed instant film. This modification resulted in a platform technology many-fold more sensitive than the former while maintaining its inherent high specificity. Preliminary data from a prototype tested during the mild 2000-2001 influenza season demonstrated that an optimized chemiluminescent test system could approach the accuracy of 14 day viral culture in a convenient 10-20 min test. This platform technology is currently being explored for the rapid detection of other pathogenic organisms where sensitivity, specificity and speed are essential in a point-of-care setting.     

A nanoparticle label/immunochromatographic electrochemical biosensor for rapid and sensitive detection of prostate-specific antigen

We present a nanoparticle (NP) label/immunochromatographic electrochemical biosensor (IEB) for rapid and sensitive detection of prostate-specific antigen (PSA) in human serum. This IEB integrates the immunochromatographic strip with the electrochemical detector for transducing quantitative signals. The NP label, made of CdSe@ZnS, serves as a signal-amplifier vehicle. A sandwich immunoreaction was performed on the immunochromatographic strip. The captured NP labels in the test zone were determined by highly sensitive stripping voltammetric measurement of the dissolved metallic component (cadmium) with a disposable-screen-printed electrode, which is embedded underneath the membrane of the test zone. Several experimental parameters (e.g., immunoreaction time, the amount of anti-PSA-NP conjugations applied) and electrochemical detection conditions (e.g., preconcentration potential and time) were optimized using this biosensor for PSA detection. The analytical performance of this biosensor was evaluated with serum PSA samples according to the “figure-of-merits” (e.g., dynamic range, reproducibility, and detection limit). The results were validated with enzyme-linked immunosorbent assay (ELISA) and showed high consistency. It is found that this biosensor is very sensitive with the detection limit of 0.02 ng mL⁻¹ PSA and is quite reproducible (with a relative standard deviation (R.S.D.) of 6.4%). This method is rapid, clinically practical, and less expensive than other diagnostic tools for PSA; therefore, this IEB coupled with a portable electrochemical analyzer shows great promise for simple, sensitive, quantitative point-of-care testing of disease-related protein biomarkers.     

To Test or to Treat? An Analysis of Influenza Testing and Antiviral Treatment Strategies Using Economic Computer Modeling

Background

Due to the unpredictable burden of pandemic influenza, the best strategy to manage testing, such as rapid or polymerase chain reaction (PCR), and antiviral medications for patients who present with influenza-like illness (ILI) is unknown.

Methodology/Principal Findings

We developed a set of computer simulation models to evaluate the potential economic value of seven strategies under seasonal and pandemic influenza conditions: (1) using clinical judgment alone to guide antiviral use, (2) using PCR to determine whether to initiate antivirals, (3) using a rapid (point-of-care) test to determine antiviral use, (4) using a combination of a point-of-care test and clinical judgment, (5) using clinical judgment and confirming the diagnosis with PCR testing, (6) treating all with antivirals, and (7) not treating anyone with antivirals. For healthy younger adults (<65 years old) presenting with ILI in a seasonal influenza scenario, strategies were only cost-effective from the societal perspective. Clinical judgment, followed by PCR and point-of-care testing, was found to be cost-effective given a high influenza probability. Doubling hospitalization risk and mortality (representing either higher risk individuals or more virulent strains) made using clinical judgment to guide antiviral decision-making cost-effective, as well as PCR testing, point-of-care testing, and point-of-care testing used in conjunction with clinical judgment. For older adults (≥65 years old), in both seasonal and pandemic influenza scenarios, employing PCR was the most cost-effective option, with the closest competitor being clinical judgment (when judgment accuracy ≥50%). Point-of-care testing plus clinical judgment was cost-effective with higher probabilities of influenza. Treating all symptomatic ILI patients with antivirals was cost-effective only in older adults.

Conclusions/Significance

Our study delineated the conditions under which different testing and antiviral strategies may be cost-effective, showing the importance of accuracy, as seen with PCR or highly sensitive clinical judgment.     

Quantum dot-based immunochromatography test strip for rapid, quantitative and sensitive detection of alpha fetoprotein

Rapid, quantitative detection of tumor markers with high sensitivity and specificity is critical to clinical diagnosis and treatment of cancer. We describe here a novel portable fluorescent biosensor that integrates quantum dot (QD) with an immunochromatography test strip (ICTS) and a home-made test strip reader for detection of tumor markers in human serum. Alpha fetoprotein (AFP), which is valuable for diagnosis of primary hepatic carcinoma, is used as a model tumor marker to demonstrate the performance of the proposed immunosensor. The principle of this sensor is on the basis of a sandwich immunoreaction that was performed on an ICTS. The fluorescence intensity of captured QD labels on the test line and control line served as signals was determined by the home-made test strip reader. The strong luminescence and robust photostability of QDs combined with the promising advantages of an ICTS and sensitive detection with the test strip reader result in good performance. Under optimal conditions, this biosensor is capable of detecting as low as 1 ng/mL AFP standard analyte in 10 min with only 50 μL sample volume. Furthermore, 1000 clinical human serum samples were tested by both the QD-based ICTS and a commercial electrochemiluminescence immunoassay AFP kit simultaneously to estimate the sensitivity, specificity and concordance of the assays. Results showed high consistency except for 24 false positive cases (false positive rate 3.92%) and 17 false negative cases (false negative rate 4.38%); the error rate was 4.10% in all. This demonstrates that the QD-based ICTS is capable of rapid, sensitive, and quantitative detection of AFP and shows a great promise for point-of-care testing of other tumor markers.     

Whole blood optical biosensor

The future of rapid point-of-care diagnostics depends on the development of cheap, noncomplex, and easily integrated systems to analyze biological samples directly from the patient (e.g. blood, urine, and saliva). A key concern in diagnostic biosensing is signal differentiation between non-specifically bound material and the specific capture of target molecules. This is a particular challenge for optical detection devices in analyzing complex biological samples. Here we demonstrate a porous silicon (PSi) label-free optical biosensor that has intrinsic size-exclusion filtering capabilities which enhances signal differentiation. We present the first demonstration of highly repeatable, specific detection of immunoglobulin G (IgG) in serum and whole blood samples over a typical physiological range using the PSi material as both a biosensor substrate and filter.     

基于杂交链式(HCR)反应的甲型流感病毒检测技术研究

甲型流感病毒的现场快速检测对于流感的及时有效防控具有重要意义．本研究基于杂交链式(HCR)反应,利用GO对荧光基团的猝灭作用及共同实现了对甲型流感病毒的快速检测．当目标序列存在时,可引发HCR反应,使短链DNA形成长链,保护FAM荧光基团不被猝灭,从而实现目标物的检测．实验结果表明,该方法在10～40 nmol/L范围内荧光强度与目标检测物浓度表现出了良好的线性关系,检测范围为5～100 nmol/L．这种HCR等温扩增检测技术具有较好的样本检测能力,具有等温、无酶、反应体系简单、操作步骤简便等优点,表现出良好的现场检测应用前景．     

Applications of commercial biosensors in clinical,food, environmental,and biothreat/biowarfare analyses

The lack of specific, low-cost, rapid, sensitive, and easy detection of biomolecules has resulted in the development of biosensor technology. Innovations in biosensor technology have enabled many biosensors to be commercialized and have enabled biomolecules to be detected onsite. Moreover, the emerging technologies of lab-on-a-chip microdevices and nanosensors offer opportunities for the development of new biosensors with much better performance. Biosensors were first introduced into the laboratory by Clark and Lyons. They developed the first glucose biosensor for laboratory conditions. Then in 1973, a glucose biosensor was commercialized by Yellow Springs Instruments. The commercial biosensors have small size and simple construction and they are ideal for point-of-care biosensing. In addition to glucose, a wide variety of metabolites such as lactate, cholesterol, and creatinine can be detected by using commercial biosensors. Like the glucose biosensors (tests) other commercial tests such as for pregnancy (hCG), Escherichia coli O157, influenza A and B viruses, Helicobacter pylori, human immunodeficiency virus, tuberculosis, and malaria have achieved success. Apart from their use in clinical analysis, commercial tests are also used in environmental (such as biochemical oxygen demand, nitrate, pesticide), food (such as glutamate, glutamine, sucrose, lactose, alcohol, ascorbic acid), and biothreat/biowarfare (Bacillus anthracis, Salmonella, Botulinum toxin) analysis. In this review, commercial biosensors in clinical, environmental, food, and biowarfare analysis are summarized and the commercial biosensors are compared in terms of their important characteristics. This is the first review in which all the commercially available tests are compiled together.     

A lateral flow biosensor for rapid detection of DNA-binding protein c-jun

A lateral flow biosensor based on an immuno-chromatographic assay has been developed for the detection of DNA-binding proteins. The biosensor is composed of four parts: a sample pad, a conjugate pad, a strip of nitrocellulose membrane and an absorbent pad. A DNA probe containing a specific protein binding consensus sequence is coated onto gold nanoparticles, while an antibody against the DNA-binding protein is immobilized onto a test zone of the nitrocellulose membrane. The target protein binds to the protein binding DNA sequence that is coated on the gold nanoparticles to form nanoparticle-DNA-protein complexes, and the complexes are then captured by the antibody immobilized on the test zone to form a red line for visual detection of the target protein. This biosensor was successfully applied to a DNA-binding protein, c-jun, and the developed biosensor allows for the rapid detection of down to 0.2 footprint unit of c-jun protein within 10 min. This biosensor was verified using HeLa cells and it visually detected c-jun activity in 100 μg of crude cell lysate protein. The antibody against c-jun used in the biosensor can distinguish c-jun from other nonspecific proteins, with high specificity.     

Antibody-induced oligomerization and activation of an engineered reporter enzyme

Our objective is to produce a protein biosensor (or molecular switch) that is specifically activated in solution by a monoclonal antibody. Many effector-dependent enzymes have evolved in nature, but the introduction of a novel regulatory mechanism into a normally unregulated enzyme poses a difficult design problem. We used site-saturation mutagenesis and screening to generate effector-activated variants of the reporter enzyme beta-glucuronidase (GUS). The specific activity of the purified epitope-tagged GUS variant was increased by up to approximately 500-fold by the addition of an equimolar concentration of a monoclonal antibody. This molecular switch is modular in design, so it can easily be re-engineered for the detection of other peptide-specific antibodies. Such antibody-activated reporters could someday enable point-of-care serological assays for the rapid detection of infectious diseases.     

Activation of nylon net and its application to a biosensor for determination of glucose in human serum

A glucose biosensor using a glucose oxidase (GO_x)-immobilized nylon net with glutaraldehyde as cross-linking reagent and an oxygen (O₂) electrode for the determination of glucose has been fabricated. The detection scheme was based on the utilization of dissolved O₂ in oxidation of glucose by the membrane bound GO_x. Crucial factors including O-alkylation temperature, reaction times of nylon net with dimethyl sulfate, l-lysine, and glutaraldehyde, and enzyme loading were examined to determine the optimal enzyme immobilization conditions for the best sensitivity of the developed glucose biosensor. In addition, the effects of pH and concentration of phosphate buffer on the response of the biosensor were studied. The glucose biosensor had a linear range of 18 μM to 1.10 mM with the detection limit of 9.0 μM (S/N = 3) and response time of 80 s. The biosensor exhibited both good operational stability with over 200 measurements and long-term storage stability. The results from this biosensor compared well with those of a commercial glucose assay kit in analyzing human serum glucose samples.     

Chemiluminescence-based biosensor for fumonisins quantitative detection in maize samples

A compact portable chemiluminescent biosensor for simple, rapid, and ultrasensitive on-site quantification of fumonisins (fumonisin B1+fumonisin B2) in maize has been developed. The biosensor integrates a competitive lateral flow immunoassay based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a contact imaging configuration. The use of chemiluminescence detection allowed accurate and objective analyte quantification, rather than qualitative or semi-quantitative information usually obtained employing conventional lateral flow immunoassays based on colloidal gold labeling. A limit of detection of 2.5 μgL(-1) for fumonisins was achieved, with an analytical working range of 2.5-500 μgL(-1) (corresponding to 25-5000 μgkg(-1) in maize flour samples, according to the extraction procedure). Total assay time was 25 min, including sample preparation. A simple and convenient extraction procedure, performed by suspending the sample in a buffered solution and rapidly heating to eliminate endogenous peroxidase enzyme activity was employed for maize flour samples analysis, obtaining recoveries in the range 90-115%, when compared with LC-MS/MS analysis. The chemiluminescence immunochromatography-based biosensor is a rapid, low cost portable test suitable for point-of-use applications.     

Rapid detection of avian influenza H5N1 virus using impedance measurement of immuno-reaction coupled with RBC amplification

Avian influenza virus (AIV) subtype H5N1 was first discovered in the 1990 s and since then its emergence has become a likely source of a global pandemic and economic loss. Currently accepted gold standard methods of influenza detection, viral culture and rRT-PCR, are time consuming, expensive and require special training and laboratory facilities. A rapid, sensitive, and specific screening method is needed for in-field or bedside testing of AI virus to effectively implement quarantines and medications. Therefore, the objective of this study was to improve the specificity and sensitivity of an impedance biosensor that has been developed for the screening of AIV H5. Three major components of the developed biosensor are immunomagnetic nanoparticles for the separation of AI virus, a microfluidic chip for sample control and an interdigitated microelectrode for impedance measurement. In this study polyclonal antibody against N1 subtype was immobilized on the surface of the microelectrode to specifically bind AIV H5N1 to generate more specific impedance signal and chicken red blood cells (RBC) were used as biolabels to attach to AIV H5N1 captured on the microelectrode to amplify impedance signal. RBC amplification was shown to increase the impedance signal change by more than 100% compared to the protocol without RBC biolabels, and was necessary for forming a linear calibration curve for the biosensor. The use of a second antibody against N1 offered much greater specificity and reliability than the previous biosensor protocol. The biosensor was able to detect AIV H5N1 at concentrations down to 10(3) EID(50)ml(-1) in less than 2h.     

合成生物学细胞传感技术在食品安全快速检测中的应用

合成生物学细胞传感技术为快速、现场检测食品污染物提供了一种新型替代方法。由于细胞内环境相对稳定,合成生物学细胞传感器有较强的抗干扰能力;由于细胞能够通过自我复制而实现增殖,细胞传感器在生产上具有简单、廉价、快速的特点,因此在食品安全快速检测中具有良好的应用前景。本文综述了合成生物学细胞传感器核心元件的组成、构建方法和类型,介绍了多功能细胞传感器的合成生物学基因回路,列举了细胞传感器在食品安全快速检测中的商业化应用前景,并阐述了细胞传感器在食品安全快速检测中的挑战和发展趋势。     

Genosensor for rapid,sensitive, specific point-of-care detection of H1N1 influenza (swine flu)

A 5′ amine group-linked haemagglutinin (HA) gene-specific probe was attached over the surface of a working electrode to develop a rapid, specific, and sensitive point of care detection assay for H1N1 (swine flu) in human respiratory nasal swabs. The probe was attached with a cysteine covered screen-printed gold electrode via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). The electrochemical assay was performed using differential pulse voltammetry with the use of the redox indicator methylene blue for the detection of different concentrations of the single-stranded viral genome. The developed genosensor showed high sensitivity for H1N1 influenza virus with a detection limit of 0.002 ng/6 μL of viral nucleic acid in the sample. Samples were analysed by quantitative real-time Polymerase Chain Reaction as well as by conventional PCR. The genosensor showed high specificity, as no cross-reaction was observed with the heterologous nucleic acid of different pathogens (Salmonella typhi, Neisseria meningitides, and Streptococcus pyogenes) and human DNA, and it was specific for H1N1 with a sensitivity of ∼49 μA cm⁻² ng^-1. Genosensor is based on a very simple methodology that can be followed based on its easy-to-access approach. It is quick and could be used as a point-of-care test for the detection of influenza virus within 30 min.     

An autonomous CMOS hysteretic sensor for the detection of desorption-free DNA hybridization

This paper describes a sensor for label-free, fully electrical detection of DNA hybridization based on capacitive changes in the electrode-electrolyte interface. The sensor measures capacitive changes in real time according to a charging-discharging principle that is limited by the hysteresis window. In addition, a novel autonomous searching technique, which exclusively monitors desorption-free hybridized electrodes among electrode arrays, enhances the performance of the sensor compared with conventional capacitive measurement. The sensor system achieves a detection range of 80 dB. The integrated circuit sensor is fabricated with a 0.35 μm CMOS process. The proposed sensor offers rapid, robust and inexpensive measurement of capacitance with highly integrated detection circuitry. It also facilitates quantitative evaluations of molecular densities on a chip with distinctive impedance variations by monitoring desorption-free hybridized electrodes. Our electrical biosensor has great potential for use with bio analytical tools and point-of-care diagnosis.     

A microbial biosensor for hydrogen sulfide monitoring based on potentiometry

In this study, a microbial biosensor for hydrogen sulfide (H₂S) detection based on Thiobacillus thioparus immobilized in a gelatin matrix was developed. The T. thioparus was immobilized via either surface adsorption on the gelatin matrix or entrapment in the matrix. The bacterial and gelatin concentration in the support were then varied in order to optimize the sensor response time and detection limit for both methods. The optimization was conducted by a statistical analysis of the measured biosensor response with various bacterial and polymer concentrations. According to our experiments with both immobilization methods, the optimized conditions for the entrapment method were found to be a gelatin concentration of 1% and an optical density of 82. For the surface adsorption method, 0.6% gelatin and an optical density of 88 were selected as the optimal conditions. A statistical model was developed based on the extent of the biosensor response in both methods of immobilization. The maximum change in the potential of the solution was 23.16 mV for the entrapment method and 34.34 mV for the surface absorption method. The response times for the entrapment and adsorption methods were 160 s and 105 s, respectively. The adsorption method is more advantageous for the development of a gas biosensor due to its shorter response time.     

Using a Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Chicken Carcass

Chicken is one of the most popular meat products in the world. Salmonella Typhimurium is a common foodbome pathogens associated with the processing of poultry. An optical Surface Plasmon Resonance （SPR） biosensor was sensitive to the presence of Salmonella Typhimurium in chicken carcass. The Spreeta biosensor kits were used to detect Salmonella Typhimurium on chicken carcass successfully. A taste sensor like electronic tongue or biosensors was used to basically ＂taste＂ the object and differentiated one object from the other with different taste sensor signatures. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with dif- ferent antisera or other molecular species. The selectivity of the SPR biosensor was assayed using a series of antibody con- centrations and dilution series of the organism. The SPR biosensor showed promising to detect the existence of Salmonella Typhimurium at 1 x 106 CFU/ml. Initial results show that the SPR biosensor has the potential for its application in pathogenic bacteria monitoring. However, more tests need to be done to confirm the detection limitation.     

用于新型冠状病毒检测的核酸等温扩增技术研究进展

核酸检测作为新型冠状病毒肺炎(COVID-19)筛查诊断和病情监测的主要手段,在疫情防控中发挥了重要作用。虽然实时荧光定量PCR被认为是新型冠状病毒(SARS-CoV-2)核酸检测的金标准,但其依赖荧光定量PCR仪且扩增检测时间较长,难以实现现场快速检测。因此许多基于核酸等温扩增的SARS-CoV-2检测方法相继诞生。等温扩增对仪器温控要求不高,通过与微流控芯片和可视化检测技术结合,可进一步简化操作、降低成本,为SARS-CoV-2现场快速筛查提供有力的技术支撑。本文围绕已报道的SARS-CoV-2等温扩增检测方法原理、检测性能及优缺点进行探讨,为进一步发展SARS-CoV-2现场快速检测平台提供参考。     

Electrochemical DNA biosensor for screening of chlorinated benzene pollutants

In this work, an electrochemical DNA biosensor based on double-stranded DNA modified Au electrode (dsDNA/Au) was proposed for the rapid screening and detection of chlorinated benzenes pollutants, in which redox-active methylene blue (MB) was used to amplify the interaction between dsDNA and the target analyte. Using hexachlorobenzene (HCB) as a model analyte of chlorinated benzenes, the biosensor demonstrated a linear response with the logarithm of HCB concentrations from 100 pmol L(-1) to 100 nmol L(-1). The obtained detection limit was 30 pmol L(-1), which was remarkably superior to other biosensors. The interaction mechanism of the biosensor with HCB was proposed based on systematical characterization by cyclic voltammetry (CV), differential pulse voltammetry (DPV), UV-vis spectrometry and electrochemical quartz crystal microbalance (EQCM). Further studies revealed that the biosensor could screen chlorinated benzenes in the presence of 100 fold amount of other co-existing chemicals (ethyl acetate and sodium oxalate, etc.), and the response signal of the biosensors for different chlorinated benzenes was correlative to their respective toxicity. The proposed biosensor proved to be a promising "alarm" tool for rapid screening of chlorinated benzenes in real water samples.     

Development of an immunochromatographic test system for the detection of human epidermal growth factor

A method was developed for the rapid detection of human epidermal growth factor based on a sandwich-format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid flow movement across the membrane components of the test strip, immunochemical reactions, and the formation of colored lines. Requirements on the configuration of the test system in order to achieve the lowest limit of detection were defined in the course of the development of the assay. It was shown that this method enables the detection of human epidermal growth factor within 5 min at concentrations as low as 10 pg/mL in aqueous solutions, urine, and the blood serum and plasma. The developed test system can be used for point-of-care diagnostics.