Paper-based electrochemical immunosensor for highly sensitive detection of chicken anemia virus.

Chicken anemia virus (CAV) is one of the primary causes of morbidity and mortality in young chickens. Given the importance of timely detection for maintaining livestock quality, there is a pressing need for rapid and field-deployable diagnostic tools. This study introduces a highly sensitive paper-based electrochemical immunosensor (PEI) for the detection of the 60 amino acid N-terminally truncated viral protein 1 (Δ60VP1), a derivative of the CAV capsid (VP1). A custom antibody was produced for precise immunoassay detection, with results obtainable within 30 min using Square Wave Voltammetry (SWV). The underlying mechanism involves an immunocomplex in the sample zone that hinders the electron transfer of redox species, thereby reducing the current signal in proportion to the Δ60VP1 concentration. Under optimal conditions, the detection linearity for Δ60VP1 ranged from 80 to 2500 ng/mL, with a limit of detection (LoD) of 25 ng/mL. This device was then successfully applied to detect VP1 in 29 chicken serum samples, achieving 91.6% sensitivity and 94.1% selectivity. In conclusion, the PEI device presents a promising solution for rapid, sensitive, and disposable detection of chicken pathogens, potentially revolutionizing productivity and quality assurance in chicken farming.

Paper-Based Laser-Pyrolyzed Electrofluidics: An Electrochemical Platform for Capillary-Driven Diagnostic Bioassays.

Microfluidic paper-based analytical devices (µPADs) are indispensable tools for disease diagnostics. The integration of electronic components into µPADs enables new device functionalities and facilitates the development of complex quantitative assays. Unfortunately, current electrode fabrication methods often hinder capillary flow, considerably restricting µPAD design architectures. Here, laser-induced graphenization is presented as an approach to fabricate porous electrodes embedded into cellulose paper. The resulting electrodes not only have high conductivity and electrochemical activity, but also retain wetting properties for capillary transport. Paper-based electrofluidics, including a lateral flow device for injection analysis of alkaline phosphatase in serum and a vertical flow device for quantitative detection of HPV16 with a CRISPR-based assay are demonstrated. It is expected that this platform will streamline the development of diagnostic devices that combine the operational simplicity of colorimetric lateral flow tests with the added benefits and possibilities offered by electronic signaling.

An amplification-free ultra-sensitive electrochemical CRISPR/Cas biosensor for drug-resistant bacteria detection.

Continued development of high-performance and cost-effective in vitro diagnostic tools is vital for improving infectious disease treatment and transmission control. For nucleic acid diagnostics, moving beyond enzyme-mediated amplification assays will be critical in reducing the time and complexity of diagnostic technologies. Further, an emerging area of threat, in which in vitro diagnostics will play an increasingly important role, is antimicrobial resistance (AMR) in bacterial infections. Herein, we present an amplification-free electrochemical CRISPR/Cas biosensor utilizing silver metallization (termed E-Si-CRISPR) to detect methicillin-resistant Staphylococcus aureus (MRSA). Using a custom-designed guide RNA (gRNA) targeting the mecA gene of MRSA, the Cas12a enzyme allows highly sensitive and specific detection when employed with silver metallization and square wave voltammetry (SWV). Our biosensor exhibits excellent analytical performance, with detection and quantitation limits of 3.5 and 10 fM, respectively, and linearity over five orders of magnitude (from 10 fM to 0.1 nM). Importantly, we observe no degradation in performance when moving from buffer to human serum samples, and achieve excellent selectivity for MRSA in human serum in the presence of other common bacteria. The E-Si-CRISPR method shows significant promise as an ultrasensitive field-deployable device for nucleic acid-based diagnostics, without requiring nucleic acid amplification. Finally, adjustment to a different disease target can be achieved by simple modification of the gRNA protospacer.

Machine learning and chemometrics for electrochemical sensors: moving forward to the future of analytical chemistry.

Electrochemical sensors and biosensors have been successfully used in a wide range of applications, but systematic optimization and nonlinear relationships have been compromised for electrode fabrication and data analysis. Machine learning and experimental designs are chemometric tools that have been proved to be useful in method development and data analysis. This minireview summarizes recent applications of machine learning and experimental designs in electroanalytical chemistry. First, experimental designs, e.g., full factorial, central composite, and Box-Behnken are discussed as systematic approaches to optimize electrode fabrication to consider the effects from individual variables and their interactions. Then, the principles of machine learning algorithms, including linear and logistic regressions, neural network, and support vector machine, are introduced. These machine learning models have been implemented to extract complex relationships between chemical structures and their electrochemical properties and to analyze complicated electrochemical data to improve calibration and analyte classification, such as in electronic tongues. Lastly, the future of machine learning and experimental designs in electrochemical sensors is outlined. These chemometric strategies will accelerate the development and enhance the performance of electrochemical devices for point-of-care diagnostics and commercialization.

Fluorometric Paper-Based, Loop-Mediated Isothermal Amplification Devices for Quantitative Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus (MRSA).

Loop-mediated isothermal amplification (LAMP) has been widely used to detect many infectious diseases. However, minor inconveniences during the steps of adding reaction ingredients and lack of simple color results hinder point-of-care detection. We therefore invented a fluorometric paper-based LAMP by incorporating LAMP reagents, including a biotinylated primer, onto a cellulose membrane paper, with a simple DNA fluorescent dye incubation that demonstrated rapid and accurate results parallel to quantitative polymerase chain reaction (qPCR) methods. This technology allows for instant paper strip detection of methicillin-resistant Staphylococcus aureus (MRSA) in the laboratory and clinical samples. MRSA represents a major public health problem as it can cause infections in different parts of the human body and yet is resistant to commonly used antibiotics. In this study, we optimized LAMP reaction ingredients and incubation conditions following a central composite design (CCD) that yielded the shortest reaction time with high sensitivity. These CCD components and conditions were used to construct the paper-based LAMP reaction by immobilizing the biotinylated primer and the rest of the LAMP reagents to produce the ready-to-use MRSA diagnostic device. Our paper-based LAMP device could detect as low as 10 ag (equivalent to 1 copy) of the MRSA gene mecA within 36-43 min, was evaluated using both laboratory (individual cultures of MRSA and non-MRSA bacteria) and clinical blood samples to be 100% specific and sensitive compared to qPCR results, and had 35 day stability under 25 °C storage. Furthermore, the color readout allows for quantitation of MRSA copies. Hence, this device is applicable for point-of-care MRSA detection.

In Situ Nucleic Acid Amplification and Ultrasensitive Colorimetric Readout in a Paper-Based Analytical Device Using Silver Nanoplates.

A rapid, highly sensitive, and quantitative colorimetric paper-based analytical device (PAD) based on silver nanoplates (AgNPls) and loop-mediated isothermal amplification (LAMP) is presented. It is shown that cauliflower-like concatemer LAMP products can mediate crystal etching of AgNPls, with a threefold signal enhancement versus linear dsDNA. Methicillin-resistant Staphylococcus aureus (MRSA), an antimicrobial resistant bacterium that poses a formidable risk with persistently high mortality, is used as a model pathogen. Due to the excellent color contrast provided by AgNPls, the PAD allows qualitative analysis by the naked eye and quantitative analysis using a smartphone camera, with detection limits down to a single copy in just 30 min, and a linear response from 1 to 104 copies (R2 = 0.994). The entire assay runs in situ on the paper surface, which drastically simplifies operation of the device. This is the first demonstration of single copy detection using a colorimetric readout, and the developed PAD shows great promise for translation into an ultrasensitive gene-based point-of-care test for any infectious disease target, via modification of the LAMP primer set.

An ultrasensitive non-noble metal colorimetric assay using starch-iodide complexation for Ochratoxin A detection.

Colorimetric sandwich-type biosensors that can both provide sensitivity competitive with fluorescence-based approaches, and leverage reagents that are cost-effective, widely available and as safe as possible, are highly sought after. Herein, we demonstrate an alternative highly-sensitive colorimetric method for paper-based sandwich-type biosensing that uses starch-iodide complexation to simplify practical biosensing using ubiquitous reagents. Targeting the mycotoxin ochratoxin A (OTA), a covalently-immobilised OTA antibody on a cellulose surface captures OTA and forms a sandwich with OTA aptamer-conjugated glucose oxidase. Adding the chromogenic reagents at an optimized concentration, a distinct blue color develops within 30 min, offering excellent contrast with the clear/white of the negative sample. With a sampling volume down to just 5 µL, the assay exhibits concentration limits of detection and quantitation of 20 and 320 pg mL-1, respectively, and a linear range from 10-1 to 105 ng mL-1 (R2 = 0.997). The method displays excellent selectivity against related mycotoxins, excellent %recovery (95-117%) and robust operation in complex matrices (beer, urine and human serum), with no significant difference versus gold-standard liquid chromatography. Along with its excellent analytical performance, this assay benefits from non-toxic and extremely cheap reagents that can be safely disposed of in the field, and presents an attractive alternative to toxic dyes and nanoparticles.

Enzyme-Assisted Nucleic Acid Detection for Infectious Disease Diagnostics: Moving toward the Point-of-Care.

Driven by complex and interconnected factors, including population growth, climate change, and geopolitics, infectious diseases represent one of the greatest healthcare challenges of the 21st century. Diagnostic technologies are the first line of defense in the fight against infectious disease, providing critical information to inform epidemiological models, track diseases, decide treatment choices, and ultimately prevent epidemics. The diagnosis of infectious disease at the genomic level using nucleic acid disease biomarkers has proven to be the most effective approach to date. Such methods rely heavily on enzymes to specifically amplify or detect nucleic acids in complex samples, and significant effort has been exerted to harness the power of enzymes for in vitro nucleic acid diagnostics. Unfortunately, significant challenges limit the potential of enzyme-assisted nucleic acid diagnostics, particularly when translating diagnostic technologies from the lab toward the point-of-use or point-of-care. Herein, we discuss the current state of the field and highlight cross-disciplinary efforts to solve the challenges associated with the successful deployment of this important class of diagnostics at or near the point-of-care.

Droplet microfluidics: from proof-of-concept to real-world utility?

Droplet microfluidics constitutes a diverse and practical tool set that enables chemical and biological experiments to be performed at high speed and with enhanced efficiency when compared to conventional instrumentation. Indeed, in recent years, droplet-based microfluidic tools have been used to excellent effect in a range of applications, including materials synthesis, single cell analysis, RNA sequencing, small molecule screening, in vitro diagnostics and tissue engineering. Our 2011 Chemical Communications Highlight Article [Chem. Commun., 2011, 47, 1936-1942] reviewed some of the most important technological developments and applications of droplet microfluidics, and identified key challenges that needed to be addressed in the short term. In the current contribution, we consider the intervening eight years, and assess the contributions that droplet-based microfluidics has made to experimental science in its broadest sense.

An Exonuclease I-Assisted Silver-Metallized Electrochemical Aptasensor for Ochratoxin A Detection.

Ochratoxin A (OTA)-a mycotoxin produced by Aspergillus and Penicillium fungi-is a carcinogen and common trace contaminant in agricultural and processed food products. As consumption is detrimental to human and animal health, regular product monitoring is vital, and highly sensitive and portable OTA sensors are necessary in many circumstances. Herein, we report an ultrasensitive, electroanalytical aptasensor for precise determination of OTA at trace levels. The sensor leverages a DNA aptamer to capture OTA and silver metallization as a signal enhancer. Exonuclease I is used to digest unbound aptamers, engendering excellent background signal suppression and sensitivity enhancements. Efficient optimization of assay conditions is achieved using central composite design (CCD), allowing rapid evaluation of both the electrode and square wave voltammetry parameter space. The sensor exhibits excellent analytical performance, with a concentration limit of detection of 0.7 pg mL-1, a limit of quantitation of 2.48 pg mL-1, and a linear dynamic range ( R2 = 0.968) of over 6 orders of magnitude (between 1 pg mL-1 and 0.1 µg mL-1). Direct comparison with ultraperformance liquid chromatography (UPLC) indicates excellent analytical performance for standard solutions ( R2 = 0.995) and spiked beer samples ( R2 = 0.993), with almost quantitative recovery and less than 5% relative standard deviation (RSD).

Voltammetric detection of carbofuran determination using screen-printed carbon electrodes modified with gold nanoparticles and graphene oxide.

Carbofuran is a highly toxic pesticide that is heavily used in agriculture due to its high effectiveness and low cost. Improved methods that are simpler and lower cost are needed for carbofuran detection in food and agricultural samples. Herein, we describe the development of a unique electrochemical method for carbofuran-phenol, which is the main hydrolysis product of carbofuran. We have successfully developed a highly accurate and precise method in a portable size using a screen-printed carbon electrode (SPCE) that is modified with graphene oxide (GO) and gold nanoparticles (AuNPs). Consequently, the developed electrode is highly sensitive to and selective for carbofuran. Using the central composite design (CCD) approach, we optimized the method for analysis parameters including the electrode surface loadings of GO and AuNPs as well as the working solution pH. The method exhibited a wide linear range of 1-250µM for analyte detection using differential pulse voltammetry (DPV) on AuNPs/GO-SPCE under the optimized conditions. The limits of detection and quantitation were 0.22 and 0.72µM, respectively. In addition, we also report the application of the method for carbofuran determination in real cucumber and rice samples. This sensitive and selective carbofuran detection method is very promising for simple and low cost analysis in real agricultural fields.

Droplet-based glucosamine sensor using gold nanoparticles and polyaniline-modified electrode.

A droplet-based electrochemical sensor for direct measurement of D-glucosamine was developed using carbon paste electrodes (CPEs) modified with gold nanoparticles (AuNPs) and polyaniline (PANI). Central composition design (CCD) was employed as a powerful method for optimization of parameters for electrode fabrication. The optimized amounts of AuNPs and PANI obtained from the response surface were determined as 300 and 3000mgL(-1), respectively. Coupled with a droplet microfluidic system, the analysis of glucosamine was performed in a high-throughput manner with a sample throughput of at least 60 samples h(-1). In addition, the adsorption of the analyte on the electrode surface was prevented due to compartmentalization in droplets. Linearity of the proposed system was found to be in the range of 0.5-5mM with a sensitivity of 7.42×10(-3)Amol(-1)Lcm(-2) and limits of detection and quantitation of 0.45 and 1.45mM, respectively. High intraday and interday (evaluated among 3 days) precisions for the detection of 50 droplets containing glucosamine were obtained with relative standard deviation less than 3%. The system was successfully used to determine the amounts of glucosamine in supplementary products with error percentage and relative standard deviation less than 3%. In addition, the amounts of glucosamine measured using the developed sensor were in good agreement with those obtained from a CE method. These indicate high accuracy and precision of the proposed system.

Graphene-polyaniline modified electrochemical droplet-based microfluidic sensor for high-throughput determination of 4-aminophenol.

We report herein the first development of graphene-polyaniline modified carbon paste electrode (G-PANI/CPE) coupled with droplet-based microfluidic sensor for high-throughput detection of 4-aminophenol (4-AP) in pharmaceutical paracetamol (PA) formulations. A simple T-junction microfluidic platform using an oil flow rate of 1.8 µL/min and an aqueous flow rate of 0.8 µL/min was used to produce aqueous testing microdroplets continuously. The microchannel was designed to extend the aqueous droplet to cover all 3 electrodes, allowing for electrochemical measurements in a single droplet. Parameters including flow rate, water fraction, and applied detection potential (Edet) were investigated to obtain optimal conditions. Using G-PANI/CPE significantly increased the current response for both cyclic voltammetric detections of ferri/ferrocyanide [Fe(CN)6](3-/4-) (10 times) and 4-AP (2 times), compared to an unmodified electrode. Using the optimized conditions in the droplet system, 4-AP in the presence of PA was selectively determined. The linear range of 4-AP was 50-500 µM (R(2) = 0.99), limit of detection (LOD, S/N = 3) was 15.68 µM, and limit of quantification (LOQ, S/N = 10) was 52.28 µM. Finally, the system was used to determine 4-AP spiked in commercial PA liquid samples and the amounts of 4-AP were found in good agreement with those obtained from the conventional capillary zone electrophoresis/UV-Visible spectrophotometry (CZE/UV-Vis). The proposed microfluidic device could be employed for a high-throughput screening (at least 60 samples h(-1)) of pharmaceutical purity requiring low sample and reagent consumption.

Electrochemical droplet-based microfluidics using chip-based carbon paste electrodes for high-throughput analysis in pharmaceutical applications.

This paper presents the first example of a pharmaceutical application of droplet-based microfluidics coupled with chronoamperometric detection using chip-based carbon paste electrodes (CPEs) for determination of dopamine (DA) and ascorbic acid (AA). Droplets were generated using an oil flow rate of 1.80 µL min(-1), whereas a flow rate of 0.80 µL min(-1) was applied to the aqueous phase, which resulted in a water fraction of 0.31. The optimum applied potential for chronoamperometric measurements in droplets was found to be 150 mV. Highly reproducible analysis of DA and AA was achieved with relative standard deviations of less than 5% for both intra-day and inter-day measurements. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 20 and 70 µM for DA and 41 and 137 µM for AA, respectively. Linearity of this method was in the ranges of 0.02-3.0mM for DA and 0.04-3.0mM for AA. This system was successfully applied to determine the amounts of DA and AA in intravenous drugs. Calibration curves of DA and AA for quantitative analysis were obtained with good linearity with R(2) values of 0.9984 and 0.9988, respectively. Compared with the labeled amounts, the measured concentrations of DA and AA obtained from this system were insignificantly different, with error percentages of less than ±3.0%, indicating a high accuracy of the developed method.