Application of Electrochemical Aptasensors toward Clinical Diagnostics, Food, and Environmental Monitoring: Review.

Aptamers are synthetic bio-receptors of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) origin selected by the systematic evolution of ligands (SELEX) process that bind a broad range of target analytes with high affinity and specificity. So far, electrochemical biosensors have come up as a simple and sensitive method to utilize aptamers as a bio-recognition element. Numerous aptamer based sensors have been developed for clinical diagnostics, food, and environmental monitoring and several other applications are under development. Aptasensors are capable of extending the limits of current analytical techniques in clinical diagnostics, food, and environmental sample analysis. However, the potential applications of aptamer based electrochemical biosensors are unlimited; current applications are observed in the areas of food toxins, clinical biomarkers, and pesticide detection. This review attempts to enumerate the most representative examples of research progress in aptamer based electrochemical biosensing principles that have been developed in recent years. Additionally, this account will discuss various current developments on aptamer-based sensors toward heavy metal detection, for various cardiac biomarkers, antibiotics detection, and also on how the aptamers can be deployed to couple with antibody-based assays as a hybrid sensing platform. Aptamers can be used in various applications, however, this account will focus on the recent advancements made toward food, environmental, and clinical diagnostic application. This review paper compares various electrochemical aptamer based sensor detection strategies that have been applied so far and used as a state of the art. As illustrated in the literature, aptamers have been utilized extensively for environmental, cancer biomarker, biomedical application, and antibiotic detection and thus have been extensively discussed in this article.

Carboxylic group riched graphene oxide based disposable electrochemical immunosensor for cancer biomarker detection.

In this work, we have developed for the first time a carboxylic group riched graphene oxide based disposable electrochemical immunosensor for cancer biomarker detection using methylene blue (MB). The developed immunosensor is highly sensitive for detection of biomarker Mucin1 (MUC1) in human serum samples. Development of this disposable electrochemical immunosensor was premeditated by applying specific monoclonal antibodies against MUC1. In this method, we explored highly conductive surface of carboxylic group (-COOH-) rich graphene oxide (GO) on screen-printed carbon electrodes (SPCE). This modified GO-COOH-SPCE was employed for the detection of MUC1 protein based on the reaction with methylene blue (MB) redox probe using differential pulse voltammetry (DPV) technique. Developed immunosensor exhibited good detection range for MUC1 with excellent linearity (0.1 U/mL- 2 U/mL), with a limit of detection of 0.04 U/mL. Upon potential application of developed biosensor, good recoveries were recorded in the range of 96-96.67% with % R.S.D 4.2. Analytical performance of the developed immunosensor assures the applicability in clinical diagnostic applications.

A Hand-Held Point-of-Care Biosensor Device for Detection of Multiple Cancer and Cardiac Disease Biomarkers Using Interdigitated Capacitive Arrays.

This paper presents a hand-held point-of-care device that incorporates a lab-on-a-chip module with interdigitated capacitive biosensors for label-free detection of multiple cancer and cardiovascular disease biomarkers. The developed prototype is comprised of a cartridge incorporating capacitive biodetection sensors, a sensitive capacitive readout electronics enclosed in a hand-held unit, and data analysis software calculating the concentration of biomarkers using previously stored reference database. The capacitive biodetection sensors are made of interdigitated circular electrodes, which are preactivated with single (for detecting one biomarker) or multiple specific antibodies (for detecting multiple disease biomarkers). Detection principle of capacitive biosensor is based on measuring the level of capacitance change between interdigitated electrode pairs induced by the change in dielectric constant due to affinity-based electron exchange in between antibodies/antigens and electrodes. The more antibody-antigens binding occurs, the more capacitance change is measured due to the change in dielectric constant of the capacitance media. The device uses preactivated ready-to-use cartridges embedded with capacitive biosensors with shelf-life of three months under optimal conditions, and is capable of onsite diagnosis and can report the result in less than 30 min. The device is verified with real patient blood samples for six different disease biomarkers.

Electrochemical Quartz Crystal Nanobalance (EQCN) Based Biosensor for Sensitive Detection of Antibiotic Residues in Milk.

An electrochemical quartz crystal nanobalance (EQCN), which provides real-time analysis of dynamic surface events, is a valuable tool for analyzing biomolecular interactions. EQCN biosensors are based on mass-sensitive measurements that can detect small mass changes caused by chemical binding to small piezoelectric crystals. Among the various biosensors, the piezoelectric biosensor is considered one of the most sensitive analytical techniques, capable of detecting antigens at picogram levels. EQCN is an effective monitoring technique for regulation of the antibiotics below the maximum residual limit (MRL). The analysis of antibiotic residues requires high sensitivity, rapidity, reliability and cost effectiveness. For analytical purposes the general approach is to take advantage of the piezoelectric effect by immobilizing a biosensing layer on top of the piezoelectric crystal. The sensing layer usually comprises a biological material such as an antibody, enzymes, or aptamers having high specificity and selectivity for the target molecule to be detected. The biosensing layer is usually functionalized using surface chemistry modifications. When these bio-functionalized quartz crystals are exposed to a particular substance of interest (e.g., a substrate, inhibitor, antigen or protein), binding interaction occurs. This causes a frequency or mass change that can be used to determine the amount of material interacted or bound. EQCN biosensors can easily be automated by using a flow injection analysis (FIA) setup coupled through automated pumps and injection valves. Such FIA-EQCN biosensors have great potential for the detection of different analytes such as antibiotic residues in various matrices such as water, waste water, and milk.

A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads.

Lysozyme (Lys) commonly presents in wines and are known to cause toxicological impact on human health. The need of highly sensitive and reliable detection methods are evident in such matrix. In this work, we developed a competitive aptamer based assay for detection of Lys by employing carboxylated magnetic beads as a support to immobilize the target molecule Lys. The used aptamer sequence was biotinylated which further binds with Streptavidin-Alkaline phosphatase (Stp-ALP) in the micro wells. Colorimetric tests were performed in order to optimize different experimental parameters. The Lys assay showed a good linearity in the range of 5-140nM with a limit of detection (LOD) 10nM. The mid-point value (IC50) 110nM and the analysis time (60min) validated the developed aptasensor as a promising tool for routine use. The assay displayed good recoveries of Lys in the range 99.00-99.27% and was demonstrated for the detection of Lys in wine samples.

Ultrasensitive detection of streptomycin using flow injection analysis-electrochemical quartz crystal nanobalance (FIA-EQCN) biosensor.

This work presents the development of an ultrasensitive biosensor for detection of streptomycin residues in milk samples using flow injection analysis-electrochemical quartz crystal nanobalance (FIA-EQCN) technique. Monoclonal antibody specific to streptomycin was immobilized on to the thiol modified gold quartz crystal surface. A broad dynamic range (0.3-300 ng/mL) was obtained for streptomycin with a good linearity in the range 0.3-10 ng/mL for PBS and 0.3-50 ng/mL for milk. The correlation coefficient (R(2)) of the biosensor was found to be 0.994 and 0.997 for PBS and milk respectively. Excellent recoveries were obtained from the streptomycin spiked milk samples in the range 98-99.33%, which shows the applicability of the developed biosensor in milk. The reproducibility of the developed biosensor was found satisfactory with % RSD (n=5) 0.351. A good co-relation was observed between the streptomycin recoveries measured through the developed biosensor and the commercial ELISA kit. The analytical figures of merit of the developed biosensor confirm that the developed FIA-EQCN biosensor could be very effective for low-level detection of streptomycin in milk samples.

Flow injection analysis biosensor for urea analysis in urine using enzyme thermistor.

There is a need for analytical methods capable of monitoring urea levels in urine for patients under clinical monitoring to appraise renal function. Herein, we present a practical method to quantify levels of urea in human urine samples using flow injection analysis-enzyme thermistor (FIA-ET) biosensor. The biosensor comprises a covalently immobilized enzyme urease (Jack bean) on aminated silica support, which selectively hydrolyzes the urea present in the sample. Under optimized conditions, the developed biosensor showed a linear response in the range of 10-1,000 mM, R (2) = 0.99, and response time of 90 s in 100 mM phosphate buffer (PB) (flow rate of 0.5 mL/min, sample volume of 0.1 mL, and pH 7.2). The urea-spiked human urine samples showed minimal matrix interference in the range of 10-1,000 mM. Recoveries were obtained (92.26-99.80 %) in the spiked urine samples. The reliability and reproducibility of the developed biosensor were found satisfactory with percent relative standard deviation (% RSD) = 0.741. The developed biosensor showed excellent operational stability up to 30 weeks with 20 % loss in original response when used continuously at room temperature. These results indicate that the developed biosensor could be very effective to detect low and high levels of urea in urine samples.

Flow injection analysis biosensor for urea analysis in adulterated milk using enzyme thermistor.

Urea in adulterated milk is one of the major health concern, it is especially harmful to pregnant women, children, and the sick. A sophisticated and reliable detection system is needed to replace current diagnostic tools for the urea in the milk. In this work, we report a flow injection analysis-enzyme thermistor (FIA-ET) bio-sensing system for monitoring of urea in adulterated milk. This biosensor was made of the covalently immobilized enzyme urease (Jack bean) on controlled pore glass (CPG) and packed into a column inside thermistor, which selectively hydrolysed the urea present in the sample. The specific heat registered from the hydrolysis of urea was found proportional to the concentration of urea present in the milk sample. The biosensor showed a linear range 1-200 mM, with % R.S.D. 0.96 for urea in 100 mM phosphate buffer, pH 7.2. Good recoveries were obtained (97.56-108.7%) for urea up to 200 mM in the spiked milk samples with % R.S.D. 0.95. In the adulterated milk, a simple filtration strategy and matrix matching technique was used to analyse urea. The response time of the sensor was evaluated for urea, which was 2 min, and it gives satisfactory output. A good comparison was observed between the urea concentrations measured through FIA-ET and the colorimetric method. These results indicate that utilizing this system could be very effective to detect low and high level of urea in adulterated milk. The immobilized urease column exhibited a good operational stability up to 180 days when used continuously at room temperature.