An integrated digital polymerase chain reaction chip for multiplexed meat adulteration detection.

Meat adulteration detection is a common concern of consumers. Here, we proposed a multiplex digital polymerase chain reaction method and a low-cost device for meat adulteration detection. Using a polydimethylsiloxane microfluidic device, polymerase chain reaction reagents could be pump-free loaded into microchambers (40 × 40 chambers) automatically. Due to the independence of multiplex fluorescence channels, deoxyribonucleic acid templates extracted from different animal species could be distinguished by one test. In this paper, we designed primers and probes for four types of meat (beef, chicken, pork, and duck) and labeled each of the four fluorescent markers (hexachlorocyclohexane [HEX], 6-carboxyfluorescein [FAM], X-rhodamine [ROX], and cyanine dyes 5 [CY5]) on the probes. Specific detection and mixed detection experiments were performed on four types of meat, realizing a limit of detection of 3 copies/µL. A mixture of four different species can be detected by four independent fluorescence channels. The quantitative capability of this method is found to meet the requirements of meat adulteration detections. This method has great potential for point-of-care testing together with portable microscopy equipment.

Enhancing affinity-based electroanalytical biosensors by integrated AC electrokinetic enrichment-A mini review.

Biosensors play a central role in moving diagnostics to being on-site or decentralized. Affinity biosensor, an important category of biosensors, has important applications in clinical diagnosis, pharmaceuticals, immunology, and other fields. Affinity biosensors rely on specific binding between target analytes and biological ligands such as antibodies, nucleic acids, or other receptors to generate measurable signals. Oftentimes the target analytes in practical samples are of low abundance in a complex matrix. Traditional affinity biosensors mainly rely on random diffusion of analytes in solution to conjugate with biorecognition elements on the sensor surface of electrodes. The process may take hours or even days, which is not conducive to rapid and sensitive detection of biosensors. Therefore, it is strongly desired to incorporate an enrichment mechanism for target analytes into biosensor-based detection. AC electrokinetic (ACEK) effect can realize rapid enrichment of analytes by application of AC electric fields, which holds great promise for achieving high sensitivity, low detection limit, and rapid turnaround. This article reviews the studies of affinity biosensors integrated with ACEK enrichment in the past decade, and summarizes the latest detection methods, detection devices and applications, hoping to provide some insights and references for researchers in related fields.

Rapid and Sensitive Detection of miRNA Based on AC Electrokinetic Capacitive Sensing for Point-of-Care Applications.

A sensitive and efficient method for microRNAs (miRNAs) detection is strongly desired by clinicians and, in recent years, the search for such a method has drawn much attention. There has been significant interest in using miRNA as biomarkers for multiple diseases and conditions in clinical diagnostics. Presently, most miRNA detection methods suffer from drawbacks, e.g., low sensitivity, long assay time, expensive equipment, trained personnel, or unsuitability for point-of-care. New methodologies are needed to overcome these limitations to allow rapid, sensitive, low-cost, easy-to-use, and portable methods for miRNA detection at the point of care. In this work, to overcome these shortcomings, we integrated capacitive sensing and alternating current electrokinetic effects to detect specific miRNA-16b molecules, as a model, with the limit of detection reaching 1.0 femto molar (fM) levels. The specificity of the sensor was verified by testing miRNA-25, which has the same length as miRNA-16b. The sensor we developed demonstrated significant improvements in sensitivity, response time and cost over other miRNA detection methods, and has application potential at point-of-care.

Recent development in chitosan nanocomposites for surface-based biosensor applications.

Recent years have witnessed ever expanding use of biosensors in the fields of environmental monitoring, homeland security, pharmaceutical, food and bioprocessing, and agricultural industries. To produce effective and reliable biosensors, good quality immobilization of biological recognition elements is critical. Chitosan and its nanocomposites emerge as an excellent immobilization matrix on biosensor surface. As a natural polysaccharide, chitosan has many useful characteristics, such as high permeability and mechanical strength, biocompatibility and non-toxicity, availability, and low cost. Due to the presence of amino and hydroxyl groups on chitosan, chitosan can easily crosslink with a variety of nanomaterials. This investigation of chitosan nanocomposite-based biosensors presents recent development and innovations in the preparation of chitosan nanocomposites in coordination with biosensors for various bio-detection applications, including chitosan nanocomposites formed with carbon nanomaterials, various inorganic and biological complexes. These chitosan nanocomposite based biosensors have demonstrated good sensitivity selectivity and stability for the detection of different types of targets ranging from glucose, proteins, DNAs, small biomolecules to bacteria. It is in our hope that this review will offer guidance for the development of novel biosensors and open up opportunities in the field of biosensor research.

A highly sensitive aptasensor for on-site detection of lipopolysaccharides in food.

A rapid, low-cost, highly sensitive, and specific capacitive aptasensor is presented for detection of lipopolysaccharides (LPS). Exposure to LPS could cause fever, gram-negative sepsis, septic shock, and eventual death. Hence, rapid, low cost, and sensitive detection of LPS is pivotal for the safety of food, pharmaceutical, and medical devices and products. In this work, a capacitive sensing method based on alternating current electrokinetics is developed to achieve rapid and specific detection of LPS. This method uses an alternating current signal for two purposes. One is to induce positive dielectrophoresis, which attracts LPS toward the sensor electrodes' surface and accelerates its binding with the immobilized aptamer probe. The other purpose is to simultaneously sense the binding reaction by measuring the interfacial capacitance change on the electrodes' surface. The testing procedures and instrumentation setup of this sensing platform are significantly simplified while finding quantitative concentrations of both analytical and complex samples within 30 s. When testing analytical samples of LPS from Escherichia coli O55:B5, a LOD of 4.93 fg/mL is achieved. The recovery analysis is also performed with LPS spiked in a complex matrix and good recovery rates are demonstrated. This work provides an affordable and field-deployable platform for highly sensitive and real-time LPS detection.

Rapid detection of trace Cu2+ using an l-cysteine based interdigitated electrode sensor integrated with AC electrokinetic enrichment.

Copper is an indispensable trace element for human health. Too much or too little intake of copper ion (Cu2+ ) can lead to its own adverse health conditions. Therefore, detection of Cu2+ is always of vital importance. In this work, a simple sensor was developed for rapid detection of trace Cu2+ in water, in which L-cysteine (Cys) as a molecular probe was self-assembled on a gold interdigital electrode to form a monolayer for specific capture of Cu2+ . The interfacial capacitance of interdigital electrode was detected to indicate the target adsorption level under an AC signal working as the excitation to induce directed movement and enrichment of Cu2+ to the electrode surface. This sensor reached a limit of detection of 4.14 fM and a satisfactory selectivity against eight other ions (Zn2+ , Hg2+ , Pb2+ , Cd2+ , Mg2+ , Fe2+ , As3+ , and As5+ ). Testing of spiked tap water was also performed, demonstrating the sensor's usability. This sensor as well as the detection method shows a great application potential in fields such as environmental monitoring and medical diagnosis.

Capacitive DNA sensor for rapid and sensitive detection of whole genome human herpesvirus-1 dsDNA in serum.

This work presents a rapid, highly sensitive, low-cost, and specific capacitive DNA sensor for detection of whole genome human herpesvirus-1 DNA. This sensor is capable of direct DNA detection with a response time of 30 s, and it can be used to test standard buffer or serum samples. The sensing approach for DNA detection is based on alternating current (AC) electrokinetics. By applying an inhomogeneous AC electric field on sensor electrodes, positive dielectrophoresis is induced to accelerate DNA hybridization. The same applied AC signal also directly measures the hybridization of target with the probe on the sensor surface. Experiments are conducted to optimize the AC signal, as well as the buffers for probe immobilization and target DNA hybridization. The assay is highly sensitive and specific, with no response to human herpesvirus-2 DNA at 5 ng/mL and a LOD of 1.0 pg/mL (6.5 copies/µL or 10.7 aM) in standard buffer. When testing the double stranded (ds) DNA spiked in human serum samples, the sensor yields a LOD of 20.0 pg/mL (129.5 copies/µL or 0.21 femtomolar (fM)) in neat serum. In this work, the target is whole genome dsDNA, consequently the test can be performed without the use of enzyme or amplification, which considerably simplifies the sensor operation and is highly suitable for point of care disease diagnosis.

One-step and real-time detection of Hg2+ in brown rice flour using a biosensor integrated with AC electrothermal enrichment.

Mercury (Hg2+) is one of the most toxic heavy metals in farm products, so rapid detection of trace Hg2+ has always been sought after with high interest. Herein, we report a biosensor to specifically recognize Hg2+ in leaching solutions of brown rice flour. This sensor is simple and of low cost, with a very short assay time of 30 s. Another merit is the ultra-low limit of detection (LOD) at fM level. In addition, the specific aptamer probe realizes a good selectivity above 105: 1 against the interferences. This sensor is developed based on an aptamer-modified gold electrode array (GEA) for capacitive sensing. Alternating current electrothermal (ACET) enrichment is induced during the AC capacitance acquirement. Thus, the enrichment and detection are coupled as a single step, and pre-concentration is needless. Owing to the sensing mechanism of solid-liquid interfacial capacitance and ACET enrichment, Hg2+ level can be sensitively and rapidly reflected. Also, the sensor has a wide linear range from 1 fM to 0.1 nM and a shelf life of 15 days. This biosensor shows advantages on overall performance, enabling easy-to-operate, real-time, and large-scale Hg2+ detection in farm products.

Detecting fa leptin receptor mutation in Zucker rats with tetra-primer amplification-refractory mutation system (ARMS)-PCR.

Due to the genetic mutation (fa) in the gene encoding for leptin receptor, homozygous Zucker rats (fa-/-) develop excessive adiposity and become an experimental animal model in obesity and metabolic-related diseases research. Based on tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), we developed a method to quickly genotype Zucker rats with a mutated fa allele from their wildtype littermates. The three genotypes are clearly discriminated on 2.0% agarose gel. Our method can be used as a reliable tool to set up and maintain the breeding colony in animal facilities as well as assign animals to control and treatment groups based on their genotypes for animal studies.

Review of Electrochemical Biosensors for Food Safety Detection.

Food safety issues are directly related to people's quality of life, so there is a need to develop efficient and reliable food contaminants' detection devices to ensure the safety and quality of food. Electrochemical biosensors have the significant advantages of miniaturization, low cost, high sensitivity, high selectivity, rapid detection, and low detection limits using small amounts of samples, which are expected to enable on-site analysis of food products. In this paper, the latest electrochemical biosensors for the detection of biological contaminants, chemical contaminants, and genetically modified crops are reviewed based on the analytes of interest, electrode materials and modification methods, electrochemical methods, and detection limits. This review shows that electrochemical biosensors are poised to provide miniaturized, specific, selective, fast detection, and high-sensitivity sensor platforms for food safety.

Electrochemical Biosensors for Circulating Tumor DNA Detection.

Early diagnosis and treatment have always been highly desired in the fight against cancer, and detection of circulating tumor DNA (ctDNA) has recently been touted as highly promising for early cancer-screening. Consequently, the detection of ctDNA in liquid biopsy is gaining much attention in the field of tumor diagnosis and treatment, which has also attracted research interest from industry. However, it is difficult to achieve low-cost, real-time, and portable measurement of ctDNA in traditional gene-detection technology. Electrochemical biosensors have become a highly promising solution to ctDNA detection due to their unique advantages such as high sensitivity, high specificity, low cost, and good portability. Therefore, this review aims to discuss the latest developments in biosensors for minimally invasive, rapid, and real-time ctDNA detection. Various ctDNA sensors are reviewed with respect to their choices of receptor probes, designs of electrodes, detection strategies, preparation of samples, and figures of merit, sorted by type of electrode surface recognition elements. The development of biosensors for the Internet of Things, point-of-care testing, big data, and big health is analyzed, with a focus on their portable, real-time, and non-destructive characteristics.

Optimization of ACEK-enhanced, PCB-based biosensor for highly sensitive and rapid detection of bisphenol a in low resource settings.

In this study, we developed a low-cost and easy-to-use capacitive biosensor employing printed-circuit-board (PCB)-based technique for electrode fabrication and a specific alternative current (AC) signal for AC Electrokinetics (ACEK) effect excitation. Fast, accurate, and highly sensitive detection and quantification of bisphenol A (BPA) was achieved. An easy characterization of the biofunctionalization process is introduced by measuring interfacial capacitance which is simple and superior to most of methods currently in use. The frequency and amplitude of the AC signal used for capacitive interrogation were optimized to achieve maximum interfacial capacitance and maximum sensitivity. To evaluate the performance of the developed biosensor, its operation was compared with in-house microfabricated and commercially available electrodes. The limit-of-detection (LOD) obtained using the PCB-based electrodes was found to be at least one order of magnitude lower than that obtained with the commercial and in-house microfabricated electrodes. The linear range for BPA detection was wide from 1 fM to 10 pM with an LOD of 109.5 aM and sample to result in 20s. The biosensor operation was validated by spike-and-recovery tests of BPA using commercial food samples. Thus, the platform has a potential as an on-site detection of bisphenol A in low-resource settings.

A disposable aptasensor based on a gold-plated coplanar electrode array for on-site and real-time determination of Cu2.

Copper ion (Cu2+) is an important cofactor for many enzymes in human body. Either excessive or deficient Cu2+ in the body may cause serious dysfunctions and diseases. So sensitive determination of Cu2+ in environmental samples is of more significance for evaluation and control of Cu2+ intake. Based on a low-cost gold-plated coplanar electrode array, a disposable aptasensor is developed with an ultra-sensitive indicator of interfacial capacitance. Modified with a specially isolated DNA aptamer for Cu2+, this sensor achieves a high selectivity of 1207: 1 against non-target ions. To realize real-time response, alternating-current electrothermal effect is integrated into the capacitance measuring process to efficiently enrich the trace Cu2+. This sensor reaches a limit of detection of 2.97 fM, with a linear range from 5.0 fM to 50 pM. The response time is only 15 s, which can meet the real-time detection requirement. On-site test of practical samples is also realized using the disposable sensor combined with a handheld inductance/capacitance/resistance meter. This sensor with its portable test system provides a cost-efficient solution for on-site, real-time and sensitive detection of Cu2+, showing great application value in environment monitoring.

Real-time, selective, and low-cost detection of trace level SARS-CoV-2 spike-protein for cold-chain food quarantine.

Due to the friendly temperature for virus survival, SARS-CoV-2 is frequently found in cold-chain foods, posing a serious threat to public health. Utilizing an interdigitated microelectrode chip modified with an antibody probe and integrating dielectrophoresis enrichment with interfacial capacitance sensing, a strategy is presented for the detection of trace level spike-protein from SARS-CoV-2. It achieves a limit of detection as low as 2.29 × 10-6 ng/mL in 20 s, with a wide linear range of 10-5-10-1 ng/mL and a selectivity of 234:1. The cost for a single test can be controlled to ~1 dollar. This strategy provides a competitive solution for real-time, sensitive, selective, and large-scale application in cold-chain food quarantine.

An on-site, highly specific immunosensor for Escherichia coli detection in field milk samples from mastitis-affected dairy cattle.

Bovine mastitis is the most economically important infectious disease in dairy industry, and Escherichia coli (E. coli) is one of the major causative pathogens. Rapid identification and quantitative detection of E. coli are of great importance for bovine mastitis control and milk quality monitoring. Capitalizing on dielectrophoresis and interphase capacitance sensing, we have developed an immunosensor for E. coli detection by modifying low cost commercial microelectrodes with an E. coli specific antibody. The limit of detection reaches as low as 775 cells/mL within a 15 s' response time, which can satisfy the requirement for on-site detection and field diagnosis of bovine mastitis. To demonstrate the sensor's specificity, tests against Staphylococcus aureus and Streptococcus uberis samples are performed showing negligible responses, and the selectivity is calculated to be 3063: 1. Furthermore, a simple pretreatment protocol is developed for on-site testing of raw milk, which only involves incubation, centrifugation and dilution steps. Then correct detection of E. coli is demonstrated for both artificially inoculated and infected field milk samples. This immunosensor and the corresponding protocol have advantages in speed, sensitivity, specificity, operability, and low cost, which make it highly promising for on-site pathogen detection of bovine mastitis.

An interdigitated microelectrode based aptasensor for real-time and ultratrace detection of four organophosphorus pesticides.

With increasing industrialization of food production, residues of organophosphorus pesticides (OPs) are more frequently found in the environment including rivers, lakes and soils. Extended exposure to OPs, even at a level below 1 nM, may lead to liver and central nervous system damages in humans and animals, while existing detection methods are not sensitive enough to detect OPs at trace levels. We presented a simple-to-use aptasensor to rapidly detect broad-spectrum OPs with high sensitivity. DNA aptamer was modified on the surface of a micro interdigitated electrode chip, and AC electrokinetics was employed to accelerate the binding of OP molecules to the aptamer probe. The sensing strategy directly measured the interfacial capacitance whose change rate was adopted as a quantitative indicator of recognition events, with a sample to result detection time of 30 s. This aptasensor had a wide linear range of (fM ~ nM), and the detection limit reached (0.24-1.67) fM for four highly-toxic OPs, with good specificity. It still showed good activity after being stored in non-refrigerated environment for at least 14 days. This aptasensor as well as the detection method offer a promising solution for on-site and real-time sensitive OP detection.

Serologic Diagnosis of Taenia Solium Cysticercosis through Linear Unmixing Analysis of Biosignals from ACEK Capacitive Sensing Method.

Cysticercosis is a parasitic infection caused by adult tapeworms, and it constantly plagues the livelihoods of people from subsistence farming communities in developing countries. Diagnosis of Cysticercosis typically requires both central nervous system imaging and serological testing. The most common methods in serological testing are Enzyme-linked Immunosorbent Assay (ELISA) and Enzyme Immuno-electrotransfer Blot (EITB). Both ELISA and EITB methods are excessively time-consuming and labor-intensive. Recent research indicates that a shorter assay time and/or higher sensitivity can be achieved by integrating alternate current electrokinetics (ACEK) with biosensing. However, the raw time-series data is very noisy and the size of the dataset is extremely small, which would bring two potential challenges. On one hand, traditional statistical methods cannot extract features robust enough for high sensitivity as well as high specificity. On the other hand, the small data size limits the usage of automatic feature extractors such as deep neural networks. In this paper, we propose a linear unmixing based approach by exploiting the possibility that the time-series biological signals can be represented as linear combinations of source signals. This paper makes distinctive contributions to the field of bio-signal by introducing the unmixing model from the image processing domain to the time-series domain. Experimental results on the classification of Cysticercosis using 123 samples demonstrate the robustness and superior performance of the linear unmixing method over other conventional classifiers in handling small datasets.

Fe-based ceramic nanocomposite membranes fabricated via e-spinning and vacuum filtration for Cd2+ ions removal.

In this work, vacuum filtered and polymer mixed e-spinning membranes (ESPMs) made from or doped with Fe-based nanomaterials were successfully fabricated to remove Cd2+ ions from a neutral aqueous solution. The used Fe-based nanomaterials including FeOOH precursor Nanowires (NWs), α-Fe2O3 NWs and Fe3O4 nanoparticles (NPs) were synthesized by elevating the hydrothermal reaction temperature from 250 °C to 500 °C or doing post-heating treatment. The adsorption results showed that vacuum filtered membranes (VFMs) overall performed a better Cd2+ ions removal behavior than e-spinning ones. Among them, VFM made from Fe3O4 NPs has the highest adsorption capacity (qt) with the adsorption amount of Cd2+ ions reaching about 29.3 mg/g within only 2 min due to the high specific surface area of NPs. Models of pseudo-first-order, pseudo-second-order and intraparticle diffusion were used to study the kinetics of Cd2+ ions removal process, and a high correlation coefficient (R2) of 0.99 was obtained when pseudo-second-order model was used. It was calculated that the equilibrium rate constant of VFM made from Fe3O4 NPs has reached about 0.28 g mg-1 min-1, much smaller than those of other membranes, which indicated a high Cd2+ ions removal efficiency.

Laser-Induced Carbon-Based Smart Flexible Sensor Array for Multiflavors Detection.

We report a flexible sensor array electronic tongue system that is fabricated on a polymer substrate by the laser direct writing process for multiflavor detection. Electronic tongue is a sensing system that is applied to detect different elements with the same sensor array. By analyzing responses from different measurement units, it enables a cross-sensitivity, namely, the ability of the system to responding to a range of different analytes in solution without specific functionalization of sensors. In this article, a six-unit sensing array system was fabricated by a laser direct writing process. Sensing units were introduced on a flexible polyamide surface. A high surface-volume ratio porous carbon structure was created by a laser-induced carbonization process, which provides stable conductive carbon electrodes with high sensitivity. Different surface treatments, such as gold plating, reduced-graphene oxide coating, and polyaniline coating, were accomplished for different measurement units. By applying principal component analysis, this sensing system shows a promising result for the detection of multiple flavors. The detection limit for each element is about 0.1 mM for NaCl and sugar solutions. Also, it is able to detect 10-4 times diluted commercial table vinegar solution, which originally contains 5% acetic acid. The detection limit is theoretically lower than the human threshold of 10 mM for NaCl and sugar. Besides, the sensing system shows a high sensitivity and selectivity for mixed elements. By mapping the data points, the sensor system could detect flavor combinations and provide a reliable prediction of analyte concentration ratios.

Highly sensitive and specific on-site detection of serum cocaine by a low cost aptasensor.

Cocaine is one of the most used illegal recreational drugs. Developing an on-site test for cocaine use detection has been a focus of research effort, since it is essential to the control and legal action against drug abuse. Currently most of cocaine detection methods are time-consuming and require special or expensive equipment, and the detection often suffers from high cross-reactivity with cocaine metabolites and relative low sensitivity with the best limit of detection reported at sub nanomolar (nM) level. In this work, an aptasensor has been developed using capacitive monitoring of sensor surface incorporating alternating current electrokinetics effects to speed up molecular transport and minimize matrix effects. The aptasensor is rapid, low cost, highly sensitive and specific as well as simple-to-use for the detection of cocaine from serum. The assay has a sample-to-result time of 30 s, a limit of detection of 7.8 fM, and a linear response for cocaine ranging from 14.5fM to 14.5pM in standard buffer, which are great improvements from other reported cocaine sensors. Special buffer is used for serum cocaine detection, and a limit of detection of 13.4 fM is experimentally demonstrated for cocaine spiked in human serum (equivalent to 1.34pM cocaine in neat serum). The specificity of the biosensor is also demonstrated with structurally similar chemicals, ecgonine ethyl ester and methylecgonidine. This biosensor shows high promise in detection of low levels of cocaine from complex matrices.