Electrochemically Controlled ATRP for Cleavage-Based Electrochemical Detection of the Prostate-Specific Antigen at Femtomolar Level Concentrations.

As a single-chain glycoprotein with endopeptidase activity, the prostate-specific antigen (PSA) is valuable as an informative serum marker in diagnosing, staging, and prognosis of prostate cancer. In this report, an electrochemical biosensor based on the target-induced cleavage of a specific peptide substrate (PSA peptide) is designed for the highly selective detection of PSA at the femtomolar level, using electrochemically controlled atom transfer radical polymerization (eATRP) as a method for signal amplification. The PSA peptides, without free carboxyl sites, are attached to the gold surface via the N-terminal cysteine residue. The target-induced cleavage of PSA peptides results in the generation of carboxyl sites, to which the alkyl halide initiator α-bromophenylacetic acid (BPAA) is linked via the Zr(IV) linkers. Subsequently, the potentiostatic eATRP of ferrocenylmethyl methacrylate (FcMMA, as the monomer) leads to the surface-initiated grafting of high-density ferrocenyl polymers. As a result, a large amount of Fc redox tags can be recruited for signal amplification, through which the limit of detection (LOD) for PSA can be down to 3.2 fM. As the recognition element, the PSA peptide is easy to synthesize, chemically and thermally stable, and low-cost. Without the necessity of enzyme or nanoparticle labels, the eATRP-based amplification method is easy to operate and low-cost. Results also show that the cleavage-based electrochemical PSA biosensor is highly selective and applicable to PSA detection in complex biological samples. In view of these merits, the integration of the eATRP-based amplification method into cleavage-based recognition is believed to hold great promise for the electrochemical detection of PSA in clinical applications.

Facile and Low-Cost SPE Modification Towards Ultra-Sensitive Organophosphorus and Carbamate Pesticide Detection in Olive Oil.

Despite the fact that a considerable amount of effort has been invested in the development of biosensors for the detection of pesticides, there is still a lack of a simple and low-cost platform that can reliably and sensitively detect their presence in real samples. Herein, an enzyme-based biosensor for the determination of both carbamate and organophosphorus pesticides is presented that is based on acetylcholinesterase (AChE) immobilized on commercially available screen-printed carbon electrodes (SPEs) modified with carbon black (CB), as a means to enhance their conductivity. Most interestingly, two different methodologies to deposit the enzyme onto the sensor surfaces were followed; strikingly different results were obtained depending on the family of pesticides under investigation. Furthermore, and towards the uniform application of the functionalization layer onto the SPEs' surfaces, the laser induced forward transfer (LIFT) technique was employed in conjunction with CB functionalization, which allowed a considerable improvement of the sensor's performance. Under the optimized conditions, the fabricated sensors can effectively detect carbofuran in a linear range from 1.1 × 10-9 to 2.3 × 10-8 mol/L, with a limit of detection equal to 0.6 × 10-9 mol/L and chlorpyrifos in a linear range from 0.7 × 10-9 up to 1.4 × 10-8 mol/L and a limit of detection 0.4 × 10-9 mol/L in buffer. The developed biosensor was also interrogated with olive oil samples, and was able to detect both pesticides at concentrations below 10 ppb, which is the maximum residue limit permitted by the European Food Safety Authority.

Fast and efficient deposition of broad range of analytes on substrates for surface enhanced Raman spectroscopy.

The majority of analytical chemistry methods requires presence of target molecules directly at a sensing surface. Diffusion of analyte from the bulk towards the sensing layer is random and might be extremely lengthy, especially in case of low concentration of molecules to be detected. Thus, even the most sensitive transducer and the most selective sensing layer are limited by the efficiency of deposition of molecules on sensing surfaces. However, rapid development of new sensing technologies is rarely accompanied by new protocols for analyte deposition. To bridge this gap, we propose a method for fast and efficient deposition of variety of molecules (e.g. proteins, dyes, drugs, biomarkers, amino acids) based on application of the alternating electric field. We show the dependence between frequency of the applied electric field, the intensity of the surface enhanced Raman spectroscopy (SERS) signal and the mobility of the studied analyte. Such correlation allows for a priori selection of parameters for any desired compound without additional optimization. Thanks to the application of the electric field, we improve SERS technique by decrease of time of deposition from 20 h to 5 min, and, at the same time, reduction of the required sample volume from 2 ml to 50 µl. Our method might be paired with number of analytical methods, as it allows for deposition of molecules on any conductive surface, or a conductive surface covered with dielectric layer.

What are the Main Sensor Methods for Quantifying Pesticides in Agricultural Activities? A Review.

In recent years, there has been an increase in pesticide use to improve crop production due to the growth of agricultural activities. Consequently, various pesticides have been present in the environment for an extended period of time. This review presents a general description of recent advances in the development of methods for the quantification of pesticides used in agricultural activities. Current advances focus on improving sensitivity and selectivity through the use of nanomaterials in both sensor assemblies and new biosensors. In this study, we summarize the electrochemical, optical, nano-colorimetric, piezoelectric, chemo-luminescent and fluorescent techniques related to the determination of agricultural pesticides. A brief description of each method and its applications, detection limit, purpose-which is to efficiently determine pesticides-cost and precision are considered. The main crops that are assessed in this study are bananas, although other fruits and vegetables contaminated with pesticides are also mentioned. While many studies have assessed biosensors for the determination of pesticides, the research in this area needs to be expanded to allow for a balance between agricultural activities and environmental protection.

Rapid methods and sensors for milk quality monitoring and spoilage detection.

Monitoring of food quality, in particular, milk quality, is critical in order to maintain food safety and human health. To guarantee quality and safety of milk products and at the same time deliver those as soon as possible, rapid analysis methods as well as sensitive, reliable, cost-effective, easy-to-use devices and systems for process control and milk spoilage detection are needed. In this paper, we review different rapid methods, sensors and commercial systems for milk spoilage and microorganism detection. The main focus lies on chemical sensors and biosensors for detection/monitoring of the well-known indicators associated with bacterial growth and milk spoilage such as changes in pH value, conductivity/impedance, adenosine triphosphate level, concentration of dissolved oxygen and produced CO2. These sensors offer several advantages, like high sensitivity, fast response time, minimal sample preparation, miniaturization and ability for real-time monitoring of milk spoilage. In addition, electronic-nose- and electronic-tongue systems for the detection of characteristic volatile and non-volatile compounds related to microbial growth and milk spoilage are described. Finally, wireless sensors and color indicators for intelligent packaging are discussed.

Muscle-on-a-chip with an on-site multiplexed biosensing system for in situ monitoring of secreted IL-6 and TNF-α.

Despite the increasing number of organs-on-a-chip that have been developed in the past decade, limited efforts have been made to integrate a sensing system for in situ continual measurements of biomarkers from three-dimensional (3D) tissues. Here, we present a custom-made integrated platform for muscle cell stimulation under fluidic conditions connected with a multiplexed high-sensitivity electrochemical sensing system for in situ monitoring. To demonstrate this, we use our system to measure the release levels and release time of interleukin 6 and tumor necrosis factor alpha in vitro by 3D muscle microtissue under electrical and biological stimulations. Our experimental design has enabled us to perform multiple time point measurements using functionalized screen-printed gold electrodes with sensitivity in the ng mL-1 range. This affordable setup is uniquely suited for monitoring factors released by 3D single cell types upon external stimulation for metabolic studies.

Emerging ultrafast nucleic acid amplification technologies for next-generation molecular diagnostics.

Over the last decade, nucleic acid amplification tests (NAATs) including polymerase chain reaction (PCR) were an indispensable methodology for diagnosing cancers, viral and bacterial infections owing to their high sensitivity and specificity. Because the NAATs can recognize and discriminate even a few copies of nucleic acid (NA) and species-specific NA sequences, NAATs have become the gold standard in a wide range of applications. However, limitations of NAAT approaches have recently become more apparent by reason of their lengthy run time, large reaction volume, and complex protocol. To meet the current demands of clinicians and biomedical researchers, new NAATs have developed to achieve ultrafast sample-to-answer protocols for the point-of-care testing (POCT). In this review, ultrafast NA-POCT platforms are discussed, outlining their NA amplification principles as well as delineating recent advances in ultrafast NAAT applications. The main focus is to provide an overview of NA-POCT platforms in regard to sample preparation of NA, NA amplification, NA detection process, interpretation of the analysis, and evaluation of the platform design. Increasing importance will be given to innovative, ultrafast amplification methods and tools which incorporate artificial intelligence (AI)-associated data analysis processes and mobile-healthcare networks. The future prospects of NA POCT platforms are promising as they allow absolute quantitation of NA in individuals which is essential to precision medicine.

Label-free detection of pepsinogen 1 and 2 by polyethylene coating Lamb microfluidic device.

Early screening of gastric cancer is a critical importance for the improvement of patients' survival rate. Here, a polyethylene coating Lamb (PE-Lamb) microfluidic device with immune layer for gastric cancer label-free detection was constructed. Two serum pepsinogen 1 (PG1) and pepsinogen 2 (PG2) biomarkers were applied to screen and predict the appearance of gastric cancer. Compared with enzyme-linked immunosorbent assay (ELISA), this method achieved a higher sensitivity and less time (40 min vs 120 min). The limit of detections (LOD) were reached 60 pg/mL for PG1 and 30 pg/mL for PG2, which have two orders of magnitude lower than traditional ELISA. The linearity coefficient indexes (R2) for PG1 and PG2 were 0.992 and 0.953 respectively, which is similar to that of ELISA. In addition, PG1 and PG2 mixed antigens sample with human serum was detected by PE-Lamb approach, and the frequency response showed high reproducibility and specificity. The results indicate that PE-lamb diagnostic technique is a novel and promising method for high-throughput screening and early diagnosis of gastric cancer.

Early detection of lung cancer biomarkers through biosensor technology: A review.

Lung cancer is undoubtedly one of the most serious health issues of the 21 st century. It is the second leading cause of cancer-related deaths in both men and women　worldwide, accounting for about 1.5 million deaths annually. Despite advances in the treatment of lung cancer with new pharmaceutical products and technological improvements, morbidity and mortality rates remains a significant challenge for the cancer biologists and oncologists. The vast majority of lung cancer patients present with advanced-stage of pathological process that ultimately leads to poor prognosis and a five-year survival rate less than 20%. Early and accurate screening and analysis using cost-effective means are urgently needed to effectively diagnose the disease, improve the survival rate or to reduce mortality and morbidity associated with lung cancer patients. Thus, the only hope for early recognition of risk factors and timely diagnosis and treatment of lung cancer is biosensors technology. Novel biosensing based diagnostics approaches for predicting metastatic risks are likely to have significant therapeutic and clinical impact in the near future. This article systematically provides a brief overview of various biosensing platforms for identification of lung cancer disease biomarkers, with a specific focus on recent advancements in electrochemical and optical biosensors, analytical performances of different biosensors, challenges and further research opportunities for routine clinical analysis.

Faecal Occult Blood Point-of-Care Tests.

BACKGROUND: Early detection of colorectal cancer decreases the risk of mortality. Faecal occult blood tests (FOBT) are recognised as a useful tool for colorectal cancer screening. These non-invasive, rapid, and easy-to-carry assays are very often used as a point-of-care test and for self-testing. On the market, there are various types of FOB tests available, including chemical and immunochromatographic tests, which are based on different detection methods and differ in their sensitivity and specificity. CONCLUSIONS: Clinicians should be aware of the causes of false-negative and false-positive test results, which can vary depending on the test. Additionally, stool sampling bias may be a source of error and must be considered by the clinician. The current FOBT methods are subject to various interfering factors; items such as proper preparation of the patient prior to testing or the clinician's knowledge of testing limitations are key in correct interpreting results. Novel technologies such as FOBT DNA tests, micro RNA tests, and biochips equipped with bacteria can indicate bleeding from the gastrointestinal tract and improve diagnostics process.

Low-Cost and Rapid-Production Microfluidic Electrochemical Double-Layer Capacitors for Fast and Sensitive Breast Cancer Diagnosis.

This technical note describes a new microfluidic sensor that combines low-cost (USD $0.97) with rapid fabrication and user-friendly, fast, sensitive, and accurate quantification of a breast cancer biomarker. The electrodes consisted of cost-effective bare stainless-steel capillaries, whose mass production is already well-established. These capillaries were used as received, without any surface modification. Microfluidic chips containing electrical double-layer capillary capacitors (µEDLC) were obtained by a cleanroom-free prototyping that allows the fabrication of dozens to hundreds of chips in 1 h. This sensor provided the successful quantification of CA 15-3, a biomarker protein for breast cancer, in serum samples from cancer patients. Antibody-anchored magnetic beads were utilized for immunocapture of the marker, and then, water was added to dilute the protein. Next, the CA 15-3 detection (<2 min) was made without using redox probes, antibody on electrode (sandwich immunoassay), or signal amplification strategies. In addition, the capacitance tests eliminated external pumping systems and precise volumetric sampling steps, as well as presented low sample volume (5 µL) and high sensitivity using bare capillaries in a new design for double-layer capacitors. The achieved limit-of-detection (92.0 µU mL-1) is lower than that of most methods reported in the literature for CA 15-3, which are based on nanostructured electrodes. The data shown in this technical note support the potential of the µEDLC toward breast cancer diagnosis even at early stages. We believe that accurate analyses using a simple sample pretreatment such as magnetic field-assisted immunocapture and cost-effective bare electrodes can be extended to quantify other cancer biomarkers and even biomolecules by changing the biorecognition element.

Rapid aptasensor capable of simply detect tumor markers based on conjugated polyelectrolytes.

In this paper, a very simple, easily-operated and universal platform is proposed for tumor marker detection. In this strategy, tumor marker-specific aptamer, which can quench the fluorescence of polyfluorene-based cationic conjugated polyelectrolytes (PFN+), are used as recognizing probes. Upon addition of tumor marker, the aptamer can be assembled into the tumor marker-aptamer complex, resulting in fluorescence recovery of PFN+ and the detection of the targets. The most widely-used tumor markers, carcinoembryonic antigen (CEA) and fetoprotein (AFP) have been chosen as the model analytes for this work. The sensing method is capable of rapidly detect target protein within 5 min without complex handling procedure and expensive instruments. Compared with previous studies, the assay presented here is really simple and avoids either conjugated polyelectrolytes (CPEs) modification or oligonucleotide labeling. This method also shows a wide detection range of 3 orders of magnitude and the detection limit is 0.316 ng/mL for CEA and 1.76 ng/mL for AFP. Furthermore, the approach requires only a convenient"mix-and-detect" procedure and offers a universal platform for the sensitive detection of any target molecule of choice according to the selected aptamer.

A label-free electrochemical biosensor for direct detection of RACK 1 by using disposable, low-cost and reproducible ITO based electrode.

In this study we designed an ultrasensitive electrochemical immunosensor for RACK 1 detection using 11-cyanoundecyltrimethoxysilane (11-CUTMS) as a immobilization matrix to immobilize biorecognition element. The used silane agent (11-CUTMS) provides a favorable platform for efficient loading of anti-RACK 1 antibody. The effective loading of the biorecognition element on the 11-CUTMS matrix was monitored by scanning electron microscopy (SEM), atomic force microscopy (AFM) images and fourier transform infrared spectroscopy (FTIR) spectra. The electrochemical characterization of the immunosensor was performed by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. Moreover, biorecognition interaction between anti-RACK1 antibodies and RACK1 antigens was monitored by using single frequency technique (SFI). The operating conditions, calibration curves obtained during optimization of experiments and reproducibility of the proposed impedimetric RACK1 biosensor are also investigated and discussed. The electrochemical immunosensor illustrated a sensitive response to RACK 1 antigen with detection limit of 10.8 fg/mL and in the linear range of 0.036-2.278 pg/mL (R2 = 0.999). Owing to high specificity, good reproducibility, long stability and reusability, the fabricated immunosensor will provide a sensitive, selective approach to RACK 1 detection. Furthermore, the practical applicability in human serum samples were investigated with a satisfactory result.

Rapid recognition of volatile organic compounds with colorimetric sensor arrays for lung cancer screening.

Volatile organic compounds (VOCs) in breath can be used as biomarkers to identify early stages of lung cancer. Herein, we report a disposable colorimetric array that has been constructed from diverse chemo-responsive colorants. Distinguishable difference maps were plotted within 4 min for specifically targeted VOCs. Through the consideration of various chemical interactions with VOCs, the arrays successfully discriminate between 20 different volatile organic compounds in breath that are related to lung cancer. VOCs were identified either with the visualized difference maps or through pattern recognition with an accuracy of at least 90%. No uncertainties or errors were observed in the hierarchical cluster analysis (HCA). Finally, good reproducibility and stability of the array was achieved against changes in humidity. Generally, this work provides fundamental support for construction of simple and rapid VOC sensors. More importantly, this approach provides a hypothesis-free array method for breath testing via VOC profiling. Therefore, this small, rapid, non-invasive, inexpensive, and visualized sensor array is a powerful and promising tool for early screening of lung cancer. Graphical abstract A disposable colorimetric array has been developed with broadly chemo-responsive dyes to incorporate various chemical interactions, through which the arrays successfully discriminate 20 VOCs that are related to lung cancer via difference maps alone or chemometrics within 4 min. The hydrophobic porous matrix provides good stability against changes in humidity.

A low-cost and miniaturized potentiostat for sensing of biomolecular species such as TNF-α by electrochemical impedance spectroscopy.

Miniaturizing potentiostats, keeping their cost low and yet preserving full measurement characteristics (e.g. bandwidth, determination of capacitive/inductive contribution to sensor's impedance and parallel screening) is still an unresolved challenge in bioelectronics. In this work, the combination of simple analogue circuitry together with powerful microcontrollers and a digital filter implementation is presented as an alternative to complex and incomplete architectures reported in the literature. A low-cost acquisition electronic system fully integrated with a biosensors platform containing eight gold working microelectrodes and integrated reference and counter electrodes was developed and validated. The manufacturing cost of the prototype was kept below 300 USD. The performance of the proposed device was benchmarked against a commercial impedance analyzer through the electrochemical analysis of a highly sensitive biosensor for the detection of tumor necrosis factor α (TNF-α) within the randomly chosen range of 266pg/mL to 666ng/mL in physiological medium (PBS). A strong correlation between the outputs of both devices was found in a critical range of frequencies (1-10Hz), and several TNF-α cytokine concentrations were properly discriminated. These results are very promising for the development of low-cost, portable and miniaturized electrochemical systems for point-of-care and environmental diagnosis.

A biolayer interferometry-based enzyme-linked aptamer sorbent assay for real-time and highly sensitive detection of PDGF-BB.

Accurate, fast and sensitive detection of disease-specific protein biomarkers, especially in blood, urine, or other bodily fluids, is an important approach to achieve early disease diagnosis. Platelet-derived growth factor-BB (PDGF-BB), a widely used biomarker, is involved in a substantial number of serious diseases, such as hepatic fibrosis, atherosclerosis, age-related macular degeneration and diabetic eye disease and is often over-expressed in human malignant tumors. Therefore, the development of sensitive and specific detection methods for PDGF-BB is of great importance for the early diagnosis of disease and assessments of patient recovery. In the current study, a biolayer interferometry-based enzyme-linked aptamer sorbent assay (BLI-ELASA) was successfully established for rapid (20-25min), high-throughput (8 or 16 samples) and real-time monitoring of PDGF-BB in clinical samples. The method exhibited a broad detection range from 0.5 to 1000ng/mL of PDGF-BB (good linear range from 0.5 to 10ng/mL), with a low detection limit of 0.08ng/mL. Moreover, BLI-ELASA was applied to the detection of PDGF-BB in spiked serum and urine samples and showed a high degree of selectivity for PDGF-BB, good reproducibility, and stability. We believe that the methodology in this work can be easily adapted to detect other biomolecules in clinical samples, including viruses, pathogens and toxins, in a rapid, sensitive, high-throughput and real-time manner.

On-chip signal amplification of magnetic bead-based immunoassay by aviating magnetic bead chains.

In this work, a Lab-on-a-Chip (LOC) platform is used to electromagnetically actuate magnetic bead chains for an enhanced immunoassay. Custom-made electromagnets generate a magnetic field to form, rotate, lift and lower the magnetic bead chains (MBCs). The cost-effective, disposable LOC platform was made with a polymer substrate and an on-chip electrochemical sensor patterned via the screen-printing process. The movement of the MBCs is controlled to improve the electrochemical signal up to 230% when detecting beta-type human chorionic gonadotropin (ß-hCG). Thus, the proposed on-chip MBC-based immunoassay is applicable for rapid, qualitative electrochemical point-of-care (POC) analysis.

Efficient label-free chemiluminescent immunosensor based on dual functional cupric oxide nanorods as peroxidase mimics.

Dual-functional cupric oxide nanorods (CuONRs) as peroxidase mimics are proposed for the development of a flow-through, label-free chemiluminescent (CL) immunosensor. Forming the basis of this cost-efficient, label-free immunoassay, CuONRs, synthesized using a simple hydrothermal method, were deposited onto epoxy-activated standard glass slides, followed by immobilization of biotinylated capture antibodies through a streptavidin bridge. The CuONRs possess excellent catalytic activity, along with high stability as a solid support. Antigens could then be introduced to the sensing system, forming large immunocomplexes that prevent CL substrate access to the surface, thereby reducing the CL signal in a concentration dependent fashion. Using carcinoembryonic antigen (CEA) as a model analyte, the proposed label-free immunosensor was able to rapidly determine CEA with a wide linear range of 0.1-60ngmL-1 and a low detection limit of 0.05ngmL-1. This nanozyme-based immunosensor is simple, sensitive, cost-efficient, and has the potential to be a very promising platform for fast and efficient biosensing applications.

A novel molecularly imprinted electrochemical sensor based on graphene quantum dots coated on hollow nickel nanospheres with high sensitivity and selectivity for the rapid determination of bisphenol S.

In this paper, a novel molecularly imprinted electrochemical sensor (MIECS) based on a glassy carbon electrode (GCE) modified with graphene quantum dots (GQDs) coated on hollow nickel nanospheres (hNiNS) for the rapid determination of bisphenol S (BPS) was proposed for the first time. HNiNS and GQDs as electrode modifications were used to enlarge the active area and electron-transport ability for amplifying the sensor signal, while molecularly imprinted polymer (MIP) film was electropolymerized by using pyrrole as monomer and BPS as template to detect BPS via cyclic voltammetry (CV). Scanning electron microscope (SEM), energy-dispersive spectrometry (EDS), CV and differential pulse voltammetry (DPV) were employed to characterize the fabricated sensor. Experimental conditions, such as molar ratio of monomer to template, electropolymerization cycles, pH, incubation time and elution time were optimized. The DPV response of the MIECS to BPS was obtained in the linear range from 0.1 to 50µM with a low limit of detection (LOD) of 0.03µM (S/N = 3) under the optimized conditions. The MIECS exhibited excellent response towards BPS with high sensitivity, selectivity, good reproducibility, and stability. In addition, the proposed MIECS was also successfully applied for the determination of BPS in the plastic samples with simple sample pretreatment.

GloSensor assay for discovery of GPCR-selective ligands.

G protein-coupled receptors (GPCRs) are modulators of almost every physiological process, and therefore, are most favorite therapeutic target for wide spectrum of diseases. Ideally, high-throughput functional assays should be implemented that allow the screening of large compound libraries in cost-effective manner to identify agonist, antagonist, and allosteric modulators in the same assay. Taking advantage of the increased understanding of the GPCR structure and signaling, several commercially available functional assays based on fluorescence or chemiluminescence detection are being used in both academia and industry. In this chapter, we provide step-by-step method and guidelines to perform cAMP measurement using GloSensor assay. Finally, we have also discussed the analysis and interpretation of results obtained using this assay by providing several examples of Gs- and Gi-coupled GPCRs.