Search

SARS-CoV-2 has mutated many times since the onset of the COVID-19 pandemic, and the omicron is currently the most dominant variant. Determining the specific strain of the virus is beneficial in providing proper care and containment of the disease. We have previously reported a novel method of counting the number of particle immunoagglutination on a paper microfluidic chip using a smartphone-based fluorescence microscope. A single-copy-level detection was demonstrated from clinical saline gargle samples. In this work, we further evaluated two different SARS-CoV-2 monoclonal antibodies to spike vs. nucleocapsid antigens for detecting omicron vs. delta and spike vs. nucleocapsid proteins. The SARS-CoV-2 monoclonal antibody to nucleocapsid proteins could distinguish omicron from delta variants and nucleocapsid from spike proteins. However, such distinction could not be found with the monoclonal antibody to spike proteins, despite the numerous mutations found in spike proteins among variants. This result may suggest a clue to the role of nucleocapsid proteins in recognizing different variants. [ABSTRACT FROM AUTHOR]

Microfluidic-based biosensors have been developed for their precise automatic reaction control. However, these biosensors require external devices that are difficult to transport and use. To overcome this disadvantage, our group made an easy-to-use, cheap, and light pumpless three-dimensional photo paper–based microfluidic analytical device (3D-μPAD; weight: 1.5 g). Unlike conventional paper-based microfluidic analytical devices, the 3D-μPAD can be used to control fluid flow in a 3D manner, thus allowing sophisticated multi-step reaction control. This device can control fluid flow speed and direction accurately using only the capillary-driven flow without an external device like a pump. The flow speed is controlled by the width of the microfluidic channel and its surface property. In addition, fluid speed control and 3D-bridge structure enable the control of fluid flow direction. Using these methods, multi-step enzyme-linked immunosorbent assay (ELISA) can be done automatically in sequence by injecting solutions (sample, washing, and enzyme's substrate) at the same time in the 3D-μPAD. All the steps can be performed in 14 min, and data can be analyzed immediately. To test this device, thioredoxin-1 (Trx-1), a biomarker of breast cancer, is used as the target. In the 3D-μPAD, it can detect 0–200 ng/mL of Trx-1, and the prepared 3D-μPAD Trx-1 sensor displays excellent selectivity. Moreover, by analyzing the concentration of Trx-1 in real patients and healthy individuals' blood serum samples using the 3D-μPAD, and comparing results to ELISA, it can be confirmed that the 3D-μPAD is a good tool for cancer diagnosis. [ABSTRACT FROM AUTHOR]

Urinary tract infections (UTIs) make up a significant proportion of the global burden of disease in vulnerable groups and tend to substantially impair the quality of life of those affected, making timely detection of UTIs a priority for public health. However, economic and societal barriers drastically reduce accessibility of traditional lab-based testing methods for critical patient groups in low-resource areas, negatively affecting their overall healthcare outcomes. As a result, cellulose-based materials such as paper and thread have garnered significant interest among researchers as substrates for so-called frugal analytical devices which leverage the material's portability and adaptability for facile and reproducible diagnoses of UTIs. Although the field may be only in its infancy, strategies aimed at commercial penetration can appreciably increase access to more healthcare options for at-risk people. In this review, we catalogue recent advances in devices that use cellulose-based materials as the primary housing or medium for UTI detection and chart out trends in the field. We also explore different modalities employed for detection, with particular emphasis on their ability to be ported onto discreet casings such as sanitary products. [ABSTRACT FROM AUTHOR]

During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP—KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3—to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3–25 nM) exhibited the lowest limits of detection (ca. μg mL−1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual's immune response can impact the interpretation of assay results. [ABSTRACT FROM AUTHOR]

As an analytical support, paper, being low cost, highly abundant, of high porosity, disposable or biodegradable, and easy to use, store, transport, and print, has excellent chemical compatibility with many applications. Since the first microfluidic paper-based analytical device (μ-PAD or lab-on-paper) was proposed, the paper-based assay has never attracted as much attention as it does now. There has recently been rapidly increasing interest in using sensitive luminescence methods, for example chemiluminescence (CL) and electrogenerated chemiluminescence (ECL), as the detection strategy for lab-on-paper devices. Because of their intrinsic characteristics, CL and ECL provide outstanding performance while retaining the simplicity, low cost, multifunctionality, versatility, flexibility, and disposability of μ-PADs. The objective of this review is to cover the development of lab-on-paper-based devices using CL and ECL detection, including fabrication of paper devices, construction of sensing interfaces, signal amplification strategies, external instruments used, and applications. We believe that lab-on-paper devices with CL and ECL detection methods will meet the diverse requirements of point-of-care diagnosis. [ABSTRACT FROM AUTHOR]

In this work, a multilayer-modified paper-based colorimetric sensing platform with improved color uniformity and intensity was developed for the sensitive and selective determination of uric acid and glucose with smartphone as signal readout. In detail, chitosan, different kinds of chromogenic reagents, and horseradish peroxidase (HRP) combined with a specific oxidase, e.g., uricase or glucose oxidase (GOD), were immoblized onto the paper substrate to form a multilayer-modified test paper. Hydrogen peroxide produced by the oxidases (uricase or GOD) reacts with the substrates (uric acid or glucose), and could oxidize the co-immoblized chromogenic reagents to form colored products with HRP as catalyst. A simple strategy by placing the test paper on top of a light-emitting diode lamp was adopted to efficiently prevent influence from the external light. The color images were recorded by the smartphone camera, and then the gray values of the color images were calculated for quantitative analysis. The developed method provided a wide linear response from 0.01 to 1.0 mM for uric acid detection and from 0.02 to 4.0 mM for glucose detection, with a limit of detection (LOD) as low as 0.003 and 0.014 mM, respectively, which was much lower than for previously reported paper-based colorimetric assays. The proposed assays were successfully applied to uric acid and glucose detection in real serum samples. Furthermore, the enhanced analytical performance of the proposed method allowed the non-invasive detection of glucose levels in tear samples, which holds great potential for point-of-care analysis.ᅟ [ABSTRACT FROM AUTHOR]

This manuscript reports on the application of chemometric methods for the development of an optimized microfluidic paper-based analytical device (μPAD). As an example, we applied chemometric methods for both device optimization and data processing of results of a colorimetric uric acid assay. Box-Behnken designs (BBD) were utilized for the optimization of the device geometry and the amount of thermal inkjet-deposited assay reagents, which affect the assay performance. Measurement outliers were detected in real time by partial least squares discriminant analysis (PLS-DA) of scanned images. The colorimetric assay mechanism is based on the on-device formation of silver nanoparticles (AgNPs) through the interaction of uric acid, ammonia, and poly(vinyl alcohol) with silver ions under mild basic conditions. The yellow color originating from visible light absorption by localized surface plasmon resonance of AgNPs can be detected by the naked eye or, more quantitatively, with a simple flat-bed scanner. Under optimized conditions, the linearity of the calibration curve ranges from 0.1-5 × 10−3 mol L−1 of uric acid with a limit of detection of 33.9 × 10−6 mol L−1 and a relative standard of deviation 4.5% (n = 3 for determination of 5.0 × 10−3 mol L−1 uric acid).A chemometrics-assisted microfluidic paper-based analytical device was developed as a low-cost and rapid platform for the determination of uric acid (UA). The detection method is based on the chemical interaction of UA, ammonia, and polyvinyl alcohol under mild basic condition with silver ions inducing formation of yellow silver nanoparticles (AgNPs). [ABSTRACT FROM AUTHOR]

Despite recent advancements in the field of microfluidic paper-based analytical devices (μPADs), a key challenge remains in developing a simple and efficient μPAD with customized imaging capabilities for antioxidant assays. In the present study, we report a facile approach for μPAD fabrication through the application of transparent nail paint leading to creation of hydrophobic barriers and well-defined channels. The resultant μPADs were then characterized through scanning electron microscopy and contact angle measurements. The resolution and functional features of the fabricated μPAD were amenable to the intended assay. The μPAD's impregnated poly(methacrylic acid) (PMAA)-coated cerium oxide (CeO2) nanoparticles oxidized the 3,3′,5,5′-tetramethylbenzidine (TMB) leading to the formation of a blue-colored charge-transfer complex. The addition of different antioxidant standard solutions resulted in a reduction in the blue color in a dose-dependent manner which could be observed visually. The color intensity of the PMAA-CeO2 nanoparticle@TMB oxidation product was inversely proportional to the antioxidant concentration and was measured using customized in-house MATLAB-based image processing software. Importantly, PMAA-CeO2 nanoparticle-based μPADs demonstrated good analytical characteristics and were able to be stored for long periods without any loss of activity. Moreover, potential interferents did not pose any threat to the colorimetric signal read-out for determination of antioxidant activity. The developed method was further applied for the assessment of antioxidant activity in a variety of tea samples and performed satisfactorily in comparison with a commonly used antioxidant detection method. Collectively, the developed μPAD-based platform holds great potential as a low-cost, convenient, portable and reliable method for pursuing various on-site antioxidant assays. [ABSTRACT FROM AUTHOR]

A simple double-layered three-dimensional (3D) microfluidic paper-based analytical device (μPAD) was designed for the simultaneous determination of six metal ions—Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II)—for the first time. The 3D μPAD was composed of two paper layers: a top pretreatment layer and a bottom colorimetric detection layer. The sample solution added to the central sample reservoir of the 3D μPAD could be automatically divided into eight flow pathways and be automatically pretreated while flowing through the pretreatment zones located in the microfluidic channels, and automatically carried out the chromogenic reactions after reaching the detection zones. Random diffusion of the chromogenic reagents was effectively prevented by transport of the pretreated sample solution to the detection zones through 3D microfluidic channels with an L-type circuitous flow route design, resulting in highly increased color uniformity and reproducibility. Combined with use of a flat LED lamp as an upward lighting source and a smartphone as a convenient detector, improved color perception, highly enhanced sensitivity, and an extended detection range were obtained. Finally, the double-layered 3D μPAD was applied to the multiplexed determination of the six metal ions in mixtures and environmental samples with satisfactory results. Detection limits as low as 0.2, 0.3, 0.1, 0.03, 0.08, and 0.04 mg/L for Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II) detection, respectively, were achieved, which are about one order of magnitude lower than obtained with previously reported μPADs for the detection of metal ions. The present 3D μPAD is simple, fast, selective, sensitive, and user-friendly, and holds great application potential for multiplexed on-site analysis. [ABSTRACT FROM AUTHOR]

In this work, fast isoelectric focusing (IEF) was successfully implemented on an open paper fluidic channel for simultaneous concentration and separation of proteins from complex matrix. With this simple device, IEF can be finished in 10 min with a resolution of 0.03 pH units and concentration factor of 10, as estimated by color model proteins by smartphone-based colorimetric detection. Fast detection of albumin from human serum and glycated hemoglobin (HBA1c) from blood cell was demonstrated. In addition, off-line identification of the model proteins from the IEF fractions with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was also shown. This PAD IEF is potentially useful either for point of care test (POCT) or biomarker analysis as a cost-effective sample pretreatment method. [ABSTRACT FROM AUTHOR]

Rapid, low-cost, and sensitive nucleic acid detection and quantification assays enabled by microfluidic paper-based analytical devices (μPADs) hold great promise for point-of-care disease diagnostics and field-based molecular tests. Through the capillary action in μPAD, flexible manipulation of nucleic acid samples can be achieved without the need for external pumps or power supplies, making it possible to fabricate highly integrated sample-to-answer devices that streamline the nucleic acid extraction, separation, concentration, amplification, and detection. To detect minute amounts of genetic materials from clinical and biological samples, it is also critical to develop sensitive signal readouts that generate physically detectable signals for in-device nucleic acid detection and/or quantification. In this review, we will focus on μPAD approaches for the facile manipulation of nucleic acids and emerging signal transduction strategies allowing sensitive and specific nucleic acid detection in μPAD. [ABSTRACT FROM AUTHOR]

A 3D paper-based microfluidic device has been developed for colorimetric determination of selected heavy metals in water samples by stacking layers of wax patterned paper and double-sided adhesive tape. It has the capability of wicking fluids and distributing microliter volumes of samples from single inlet into affrays of detection zones without external pumps, thus a range of metal assays can be simply and inexpensively performed. We demonstrate a prototype of four sample inlets for up to four heavy metal assays each, with detection limits as follows: Cu (II) = 0.29 ppm, Ni(II) = 0.33 ppm, Cd (II) = 0.19 ppm, and Cr (VI) = 0.35 ppm, which provided quantitative data that were in agreement with values gained from atomic absorption. It has the ability to identify these four metals in mixtures and is immune to interferences from either nontoxic metal ions such as Na(I) and K(I) or components found in reservoir or beach water. With the incorporation of a portable detector, a camera mobile phone, this 3D paper-based microfluidic device should be useful as a simple, rapid, and on-site screening approach of heavy metals in aquatic environments. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

We describe the use of a chemometrics-based computational platform to optimize a glucose assay on a microfluidic paper-based analytical device (μPAD). Glucose is colorimetrically detected in the presence of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI). Using a Y-shaped paper microfluidic chip, the concentration of glucose, volume of reagents, and the length and width of the microfluidic channel were examined. The responses of the microfluidic chips were analyzed at the halfway point of the channel length. Variables affecting the response were screened by using a 2 factorial design, and among them, volume and concentration of the glucose were optimized by applying a rotatable central composite design (CCD). The optimum and experimental responses are 151.58 and 149.80, respectively, with an absolute error of 1.2%. [ABSTRACT FROM AUTHOR]

A disposable electrochemical lateral flow paper-based sensing device for heavy metal detection is proposed. The quantification of lead and cadmium in aqueous samples is demonstrated in a range from 10 to 100 ppb with a limit of detection of 7 and 11 ppb respectively. Moreover, the platform itself, which is made of paper, served as a sample pretreatment material due to its filtering properties. Real samples, especially in which the matrix is usually turbid and would in principle need a previous filtration, are successfully analyzed. This lab-on-paper device is simple, low cost, easy to fabricate, and portable, being a promising tool for new point-of-care applications in environmental monitoring, public health, and food safety. [ABSTRACT FROM AUTHOR]

This article describes the use of microfluidic paper-based analytical devices (μPADs) to perform quantitative chemical assays with internal standards. MicroPADs are well-suited for colorimetric biochemical assays; however, errors can be introduced from the background color of the paper due to batch difference and age, and from color measurement devices. To reduce errors from these sources, a series of standard analyte solutions and the sample solution are assayed on a single device with multiple detection zones simultaneously; an analyte concentration calibration curve can thus be established from the standards. Since the μPAD design allows the colorimetric measurements of the standards and the sample to be conducted simultaneously and under the same condition, errors from the above sources can be minimized. The analytical approach reported in this work shows that μPADs can perform quantitative chemical analysis at very low cost. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

This paper reports on an inkjet printing method for the fabrication of lateral flow immunochromatographic devices made from a single piece of filter paper by patterning microfluidic channels and dispensing immunosensing inks, requiring only a single printing apparatus. This “paperfluidic” immunosensing device allows for a less time-consuming and more low-cost fabrication compared with the conventional immunochromatographic strips requiring multiple pads, plastic or nylon backing, and a plastic case. A sandwich immunoreaction was performed on the patterned immunosensing paper device, and the sensitivity of the device was optimized with an IgG model analyte. Inkjet-printed antibodies on the test line and the control line were immobilized by physical adsorption, resulting in a very simple fabrication method applicable for pure cellulose surfaces. The color intensity in the test line and the control line was determined both by naked eye and by means of a color scanner in combination with a simple computer program. With the resulting paperfluidic immunosensing device, human IgG concentrations at least down to 10 μg/l could be detected within 20 min. Additionally, in order to demonstrate the feasibility of a total multianalyte sensing system, a combined immuno-chemical sensing device was also fabricated by patterning an additional microfluidic channel for a chemical assay onto the same paper substrate. This low-cost multianalyte paperfluidic sensing device thus demonstrates the feasibility of simple, portable, and disposable tools for pathogen detection in the field of medical, environmental, and food analyses, possibly resulting in useful devices in remote settings and less-industrialized countries. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Microbial resistance to currently available antibiotics poses a great threat in the global fight against infections. An important step in determining bacterial antibiotic resistance can be selective DNA sequence capture and fluorescence labeling. In this paper, we demonstrate the fabrication of simple, robust, inexpensive microfluidic devices for DNA capture and fluorescence detection of a model antibiotic resistance gene sequence. We laser micromachined polymethyl methacrylate microchannels and enclosed them using pressure-sensitive adhesive tapes. We then formed porous polymer monoliths with DNA capture probes in these microchannels and used them for sequence-specific capture, fluorescent labeling, and laser-induced fluorescence detection of picomolar (pM) concentrations of synthetic and plasmid antibiotic resistance gene targets. The relative fluorescence for the elution peaks increased with loaded target DNA concentration. We observed higher fluorescence signal and percent recovery for synthetic target DNA compared to plasmid DNA at the same loaded target concentration. A non-target gene was used for control experiments and produced < 3% capture relative to the same concentration of target. The full analysis process including device fabrication was completed in less than 90 min with a limit of detection of 30 pM. The simplicity of device fabrication and good DNA capture selectivity demonstrated herein have potential for application with processes for bacterial plasmid DNA extraction and single-particle counting to facilitate determination of antibiotic susceptibility. [ABSTRACT FROM AUTHOR]

Planar monolayer microreactor arrays (PMMRAs) make droplet-based numerical measurements and statistical analysis cheap and easy. However, PMMRAs are typically produced in complex microfluidic devices and, moreover, still requires stringent control to reduce droplet loss during heating. In this paper, a simple, reliable, and flexible method for fabricating PMMRAs in a 96-well plate is described in detail by using simple materials and low-cost equipment. The partitioned droplets spontaneously assemble into PMMRAs in the plates, and this distribution is maintained even after incubation. This is advantageous for in situ analysis based on an individual droplet in droplet digital loop-mediated isothermal amplification (ddLAMP) and does not require the transfer of positive droplets. Precise and reproducible quantification of classical swine fever virus (CSFV) extracts was executed in these PMMRAs to verify its availability. Our results demonstrate that the proposed approach not only provides a flexible and controllable execution scheme for droplet-based nucleic acid quantification in resource-limited laboratories but also opens new perspectives for numerous analytical and biochemical applications using droplets as versatile plastic microreactors. [ABSTRACT FROM AUTHOR]

We present an insert-based approach to fabricate scalable and multiplexable microfluidic devices for 3D cell culture and integration with downstream detection modules. Laser-cut inserts with a layer of electrospun fibers are used as a scaffold for 3D cell culture, with the inserts being easily assembled in a 3D-printed fluidic device for flow-based studies. With this approach, the number and types of cells (on the inserts) in one fluidic device can be customized. Moreover, after an investigation (i.e., stimulation) under flowing conditions, the cell-laden inserts can be removed easily for subsequent studies including imaging and cell lysis. In this paper, we first discuss the fabrication of the device and characterization of the fibrous inserts. Two device designs containing two (channel width = 260 μm) and four (channel width = 180 μm) inserts, respectively, were used for different experiments in this study. Cell adhesion on the inserts with flowing media through the device was tested by culturing endothelial cells. Macrophages were cultured and stimulated under different conditions, the results of which indicate that the fibrous scaffolds under flow conditions result in dramatic effects on the amount and kinetics of TNF-α production (after LPS stimulation). Finally, we show that the cell module can be integrated with a downstream absorbance detection scheme. Overall, this technology represents a new and versatile way to culture cells in a more in vivo fashion for in vitro studies with online detection modules.This paper describes an insert-based microfluidic device for 3D cell culture that can be easily scaled, multiplexed, and integrated with downstream analytical modules. [ABSTRACT FROM AUTHOR]

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl, and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. [ABSTRACT FROM AUTHOR]

In this paper, we demonstrate a rapid analysis procedure for use with a small set of rapidly mutating Y chromosomal short tandem repeat (Y-STR) loci that combines both rapid polymerase chain reaction (PCR) and microfluidic separation elements. The procedure involves a high-speed polymerase and a rapid cycling protocol to permit PCR amplification in 16 min. The resultant amplified sample is next analysed using a short 1.8-cm microfluidic electrophoresis system that permits a four-locus Y-STR genotype to be produced in 80 s. The entire procedure takes less than 25 min from sample collection to result. This paper describes the rapid amplification protocol as well as studies of the reproducibility and sensitivity of the procedure and its optimisation. The amplification process utilises a small high-speed thermocycler, microfluidic device and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The four loci used for the multiplex were selected due to their rapid mutation rates and should proved useful in preliminary screening of samples and suspects. Overall, this technique provides a method for rapid sample screening of suspect and crime scene samples in forensic casework. [ABSTRACT FROM AUTHOR]

The growing need for biological information at the single cell level has driven the development of improved cytometry technologies. Flow cytometry is a particularly powerful method that has evolved over the past few decades. Flow cytometers have become essential instruments in biomedical research and routine clinical tests for disease diagnosis, prognosis, and treatment monitoring. However, the increasing number of cellular parameters unveiled by genomic, proteomic, and metabolomic data platforms demands an augmented multiplexability. Also, the need for identification and quantification of relevant biomarkers at low levels requires outstanding analytical sensitivity and reliability. In addition, growing awareness of the advantages associated with miniaturization of analytical devices is pushing forward the progress in integrated and compact, microfluidic-based devices at the point-of-care. In this context, novel types of flow cytometers are emerging during the search to tackle these challenges. Notwithstanding the relevance of other promising alternatives to standard optical flow cytometry (e.g., mass cytometry, various optical and electrical microcytometers), this report focuses on a recent microcytometric technology based on magnetic sensors and magnetic particles integrated into microfluidic structures for dynamic bioanalysis of fluid samples—magnetic flow cytometry. Its concept, main developments, targeted applications, as well as the challenges and trends behind this technology are presented and discussed. Graphical abstract ᅟ "Kindly advise whether there is online abstract figure for this paper. If so, kindly resupply.The graphical abstract is correctly supplied. [ABSTRACT FROM AUTHOR]

Evaluation and diagnosis of blood alterations is a common request for clinical laboratories, requiring a complex technological approach and dedication of health resources. In this paper, we present a microfluidic device that owing to a novel combination of hydrodynamic and dielectrophoretic techniques can separate plasma from fresh blood in a microfluidic channel and for the first time allows optical real-time monitoring of the components of plasma without pre- or post-processing. The microchannel is based on a set of dead-end branches at each side and is initially filled using capillary forces with a 2-μL droplet of fresh blood. During this process, stagnation zones are generated at the dead-end branches and some red blood cells (RBCs) are trapped there. An electric field is then applied and dielectrophoretic trapping of RBCs is used to prevent more RBCs entering into the channel, which works like a sieve. Besides, an electroosmotic flow is generated to sweep the rest of the RBCs from the central part of the channel. Consequently, an RBC-free zone of plasma is formed in the middle of the channel, allowing real-time monitoring of the platelet behavior. To study the generation of stagnation zones and to ensure RBC trapping in the initial constrictions, two numerical models were solved. The proposed experimental design separates up to 0.1 μL blood plasma from a 2-μL fresh human blood droplet. In this study, a plasma purity of 99 % was achieved after 7 min, according to the measurements taken by image analysis. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Miniaturized synthesis of positron emission tomography (PET) tracers is poised to offer numerous advantages including reduced tracer production costs and increased availability of diverse tracers. While many steps of the tracer production process have been miniaturized, there has been relatively little development of microscale systems for the quality control (QC) testing process that is required by regulatory agencies to ensure purity, identity, and biological safety of the radiotracer before use in human subjects. Every batch must be tested, and in contrast with ordinary pharmaceuticals, the whole set of tests of radiopharmaceuticals must be completed within a short-period of time to minimize losses due to radioactive decay. By replacing conventional techniques with microscale analytical ones, it may be possible to significantly reduce instrument cost, conserve lab space, shorten analysis times, and streamline this aspect of PET tracer production. We focus in this work on miniaturizing the subset of QC tests for chemical identity and purity. These tests generally require high-resolution chromatographic separation prior to detection to enable the approach to be applied to many different tracers (and their impurities), and have not yet, to the best of our knowledge, been tackled in microfluidic systems. Toward this end, we previously explored the feasibility of using the technique of capillary electrophoresis (CE) as a replacement for the “gold standard” approach of using high-performance liquid chromatography (HPLC) since CE offers similar separating power, flexibility, and sensitivity, but can readily be implemented in a microchip format. Using a conventional CE system, we previously demonstrated the successful separation of non-radioactive version of a clinical PET tracer, 3′-deoxy-3′-fluorothymidine (FLT), from its known by-products, and the separation of the PET tracer 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-cytosine (D-FAC) from its α-isomer, with sensitivity nearly as good as HPLC. Building on this feasibility study, in this paper, we describe the first effort to miniaturize the chemical identity and purity tests by using microchip electrophoresis (MCE). The fully automated proof-of-concept system comprises a chip for sample injection, a separation capillary, and an optical detection chip. Using the same model compound (FLT and its known by-products), we demonstrate that samples can be injected, separated, and detected, and show the potential to match the performance of HPLC. Addition of a radiation detector in the future would enable analysis of radiochemical identity and purity in the same device. We envision that eventually this MCE method could be combined with other miniaturized QC tests into a compact integrated system for automated routine QC testing of radiopharmaceuticals in the future.Miniaturized quality control (QC) testing of batches of radiopharmaceuticals via microfluidic analysis. The proof-of-concept hybrid microchip electrophoresis (MCE) device demonstrated the feasibility of achieving comparable performance to conventional analytical instruments (HPLC or CE) for chemical purity testing. [ABSTRACT FROM AUTHOR]

Blood is one of the most important biofluids used for clinical diagnostics. Cells and proteins in the blood can provide a rich source of information for the evaluation of human health. Efficient separation of blood components is a necessary process in order to minimize the interference of unwanted components during sensing, separation, and detection. In this paper, an insulator-based gradient dielectrophoretic device has been applied to separate red blood cells from model protein biomarkers for myocardial infarction in buffer. Within one min, red blood cells are largely depleted regardless of the minimum adherence on the channel wall. Considering the adhered red blood cells will not be transported further, a purified protein solution can be delivered for potential downstream processing or detection. [ABSTRACT FROM AUTHOR]

This paper presents a microfluidic chip for highly efficient separation of red blood cells (RBCs) from whole blood on the basis of their native magnetic properties. The glass chip was fabricated by photolithography and thermal bonding. It consisted of one inlet and three outlets, and a nickel wire of 69-μm diameter was positioned in the center of a separation channel with 149-μm top width and 73-μm depth by two parallel ridges (about 10 μm high). The two ridges were formed simultaneously during the wet etching of the channels. The nickel wire for generating the magnetic gradient inside the separation channel was introduced from the side of the chip through a guide channel. The external magnetic field was applied by a permanent magnet of 0.3 T placed by the side of the chip and parallel to the main separation channel. The RBCs were separated continuously from the 1:40 (v/v) diluted blood sample at a flow rate in the range 0.12–0.92 μL/min (9–74 mm/min) with the chip, and up to 93.7% of the RBCs were collected in the middle outlet under a flow rate of 0.23 μL/min. The cell sedimentation was alleviated by adjusting the specific density of the supporting media with bovine serum albumin. Quantum dot labeling was introduced for visual fluorescence tracking of the separation process. The uneven distribution phenomenon of the blood cells around the nickel wire was reported and discussed. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

This article reviews advances over the last five years in microfluidics and microchip-electrophoresis techniques for detection of clinical biomarkers. The variety of advantages of miniaturization compared with conventional benchtop methods for detecting biomarkers has resulted in increased interest in developing cheap, fast, and sensitive techniques. We discuss the development of applications of microfluidics and microchip electrophoresis for analysis of different clinical samples for pathogen identification, personalized medicine, and biomarker detection. We emphasize the advantages of microfluidic techniques over conventional methods, which make them attractive future diagnostic tools. We also discuss the versatility and adaptability of this technology for analysis of a variety of biomarkers, including lipids, small molecules, carbohydrates, nucleic acids, proteins, and cells. Finally, we conclude with a discussion of aspects that need to be improved to move this technology towards routine clinical and point-of-care applications. [ABSTRACT FROM AUTHOR]

The concept and realization of microfluidic total analysis systems (microTAS) have revolutionized the analytical process by integrating the whole breadth of analytical techniques into miniaturized systems. Paramount for efficient and competitive microTAS are integrated detection strategies, which lead to low limits of detection while reducing the sample volume. The concept of electrochemiluminescence (ECL) has been intriguing ever since its introduction based on Ru(bpy)32+ by Tokel and Bard [1] (J Am Chem Soc 1853:2862–2863, 1972), especially because of its immense sensitivity, nonexistent auto-luminescent background signal, and simplicity in experimental design. Therefore, integrating ECL detection into microTAS is a logical consequence to achieve simple, yet highly sensitive, sensors. However, published microanalytical devices employing ECL detection focus in general on traditional ECL chemistry and have yet to take advantage of advances made in standard bench-top ECL strategies. This review will therefore focus on the most recent advancements in microfluidic ECL approaches, but also evaluate the potential impact of bench-top ECL research progress that would further improve performance and lower limits of detection of micro analytical ECL systems, ensuring their desirability as detection principle for microTAS applications. [ABSTRACT FROM AUTHOR]

Carcinogen contaminations in the food chain, for example heavy metal ions, pesticides, acrylamide, and mycotoxins, have caused serious health problems. A major objective of food-safety research is the identification and prevention of exposure to these carcinogens, because of their impossible-to-reverse tumorigenic effects. However, carcinogen detection is difficult because of their trace-level presence in food. Thus, reliable and accurate separation and determination methods are essential to protect food safety and human health. This paper summarizes the state of the art in separation and determination methods for analyzing carcinogen contamination, especially the advances in biosensing methods. Furthermore, the application of promising technology including nanomaterials, imprinted polymers, and microdevices is detailed. Challenges and perspectives are also discussed. [ABSTRACT FROM AUTHOR]

Digital single-cell assays hold high potentials for the analysis of cell apoptosis and the evaluation of chemotherapeutic reagents for cancer therapy. In this paper, a microfluidic hydrodynamic trapping system was developed for digital single-cell assays with the capability of monitoring cellular dynamics over time. The microfluidic chip was designed with arrays of bypass structures for trapping individual cells without the need for surface modification, external electric force, or robotic equipment. After optimization of the bypass structure by both numerical simulations and experiments, a single-cell trapping efficiency of ∼90 % was achieved. We demonstrated the method as a digital single-cell assay for the evaluation of five clinically established chemotherapeutic reagents. As a result, the half maximal inhibitory concentration (IC) values of these compounds could be conveniently determined. We further modeled the gradual decrease of active drugs over time which was often observed in vivo after an injection to investigate cell apoptosis against chemotherapeutic reagents. The developed method provided a valuable means for cell apoptotic analysis and evaluation of anticancer drugs. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

This paper describes a temperature-controllable bead affinity chromatography (BAC) in a microsystem for biomarker detection, and preparing samples for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Cancer marker proteins were captured in the microsystem by BAC with RNA aptamer-immobilized microbeads. The captured proteins were then denatured and released from the microbeads by controlling temperature. The microsystem consists of a microreactor for trapping microbeads and a temperature control unit for thermal treatment of the trapped beads. We used polymethylsilxoane or single crystalline silicon in fabricating two different types of reaction chamber to compare the differences in performance originated from the materials. Carcinoembryonic antigen was concentrated and purified from human serum using the microsystem and detected by MALDI-TOF MS to demonstrate the usefulness of the microsystem. The microsystem simplifies a sample preparation process required for protein analysis and cancer biomarker detection, which will accelerate the process of cancer research. [ABSTRACT FROM AUTHOR]

In the work discussed in this paper, the effect of a high surface-to-volume ratio of a microfluidic detection cell on fluorescence quenching was studied. It was found that modification of the geometry of a microchannel can provide a wider linear range. This is a phenomenon which should be taken into consideration when microfluidic systems with fluorescence detection are developed. The dependence of the linear range for fluorescein on the surface-to-volume ratio was determined. Both fluorescence inner-filter effects and concentration self-quenching were taken into consideration. It was found that inner-filter effects have little effect on the extent of the linear range on the microscale. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems. [ABSTRACT FROM AUTHOR]

By combining cell technology and microchip technology, innovative cellular biochemical tools can be created from the microscale to the nanoscale for both practical applications and fundamental research. On the microscale level, novel practical applications taking advantage of the unique capabilities of microfluidics have been accelerated in clinical diagnosis, food safety, environmental monitoring, and drug discovery. On the other hand, one important trend of this field is further downscaling of feature size to the 10-10 nm scale, which we call extended-nano space. Extended-nano space technology is leading to the creation of innovative nanofluidic cellular and biochemical tools for analysis of single cells at the single-molecule level. As a pioneering group in this field, we focus not only on the development of practical applications of cellular microchip devices but also on fundamental research to initiate new possibilities in the field. In this paper, we review our recent progress on tissue reconstruction, routine cell-based assays on microchip systems, and preliminary fundamental method for single-cell analysis at the single-molecule level with integration of the burgeoning technologies of extended-nano space. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The control of hydrodynamic focusing in a microchannel has inspired new approaches for microfluidic mixing, separations, sensors, cell analysis, and microfabrication. Achieving a flat interface between the focusing and focused fluids is dependent on Reynolds number and device geometry, and many hydrodynamic focusing systems can benefit from this understanding. For applications where a specific cross-sectional shape is desired for the focused flow, advection generated by grooved structures in the channel walls can be used to define the shape of the focused flow. Relative flow rates of the focused flow and focusing streams can be manipulated to control the cross-sectional area of the focused flows. This paper discusses the principles for defining the shape of the interface between the focused and focusing fluids and provides examples from our lab that use hydrodynamic focusing for impedance-based sensors, flow cytometry, and microfabrication to illustrate the breadth of opportunities for introducing new capabilities into microfluidic systems. We evaluate each example for the advantages and limitations integral to utilization of hydrodynamic focusing for that particular application. [ABSTRACT FROM AUTHOR]

Capillary electrophoretic separations performed in short capillaries under high field strengths have recently emerged as a promising alternative to chip-based separations. However, the injection and detection approaches have to be adapted appropriately to enable high-throughput determinations. This paper addresses current challenges and trends in this field of research. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

In this paper we describe plasma stencilling techniques for patterning 10 mammalian cell lines on hydrophobic and cell repellent poly(dimethylsiloxane) (PDMS), methylated glass and bacterial grade polystyrene surfaces. An air plasma produced with a Tesla generator operating at atmospheric pressure was used with microengineered stencils for patterned surface oxidation, selectively transforming the surface to a hydrophilic state to enable cell adhesion and growth. Plasma stencilling obviates the need for directly patterning cell adhesion molecules. Instead, during cell culture, adhesion proteins from the media assemble in a bioactive form on the hydrophilic regions. Critically, the removal of protein patterning prior to cell culture provides the option to also use PDMS–PDMS plasma bonding to incorporate cell patterns within microfluidic systems. Linear patterns were generated using PDMS microchannel stencils, and polyimide stencils with through holes were used for the production of cellular arrays. For the production of smaller cellular arrays, a novel microcapillary-based dielectric barrier discharge system was developed. A numerical method to characterise the cell patterns is also introduced and was used to demonstrate that plasma stencilling is highly effective, with complete patterns confined during long term cell culture (>10 days). In summary, plasma stencilling is simple, rapid, inexpensive, reproducible and a potentially universal cell line patterning capability. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Magnetism-based microsystems, as those dedicated to immunoaffinity separations or (bio)chemical reactions, take benefit of the large surface area-to-volume ratio provided by the immobilized magnetic beads, thus increasing the sensitivity of the analysis. As the sensitivity is directly linked to the efficiency of the magnetic bead capture, this paper presents a simple method to enhance the capture in a microchannel. Considering a microchannel surrounded by two rectangular permanent magnets of different length ( Lm = 2, 5, 10 mm) placed in attraction, it is shown that the amount of trapped beads is limited by the magnetic forces mainly located at the magnet edges. To overcome this limitation, a polyethylene terephthalate (PET) microchip with an integrated magnetic track array has been prototyped by laser photo-ablation. The magnetic force is therefore distributed all along the magnet length. It results in a multi-plug bead capture, observed by microscope imaging, with a magnetic force value locally enhanced. The relative amount of beads, and so the specific binding surface for further immunoassays, presents a significant increase of 300% for the largest magnets. The influence of the track geometry and relative permeability on the magnetic force was studied by numerical simulations, for the microchip operating with 2-mm-long magnets. [ABSTRACT FROM AUTHOR]

Biotechnology process development involves strain testing and improvement steps aimed at increasing yields and productivity. This necessitates the high-throughput screening of many potential strain candidates, a task currently mainly performed in shake flasks or microtiter plates. However, these methods have some drawbacks, such as the low data density (usually only end-point measurements) and the lack of control over cultivation conditions in standard shake flasks. Microbioreactors can offer the flexibility and controllability of bench-scale reactors and thus deliver results that are more comparable to large-scale fermentations, but with the additional advantages of small size, availability of online cultivation data and the potential for automation. Current microbioreactor technology is analyzed in this review paper, focusing on its industrial applicability, and directions for future research are presented. [ABSTRACT FROM AUTHOR]

Demand is growing in the field of medical diagnostics for simple, disposable devices that also demonstrate fast response times, are easy to handle, are cost-efficient, and are suitable for mass production. Polymer-based microfluidic devices meet the requirements of cost efficiency and mass production and they are suitable for biosensor applications. Conducting polymer-based electrochemical sensors have shown numerous advantages in a number of areas related to human health, such as the diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology, due to their simplicity and high sensitivity. One of the most promising group of conductive polymers is poly(3,4-ethylenedioxythiophene) (PEDOT) and its derivatives due to their attractive properties: high stability, high conductivity (up to 400–600 S/cm) and high transparency. This review paper summarizes newly developed methods associated with the application of PEDOT to diagnostic sensing. [ABSTRACT FROM AUTHOR]

In this paper, we successfully separated malignant human breast cancer epithelial cells (MCF 7) from healthy breast cells (MCF 10A) and analyzed the main parameters that influence the separation efficiency with an advanced dielectrophoresis (DEP)-activated cell sorter (DACS). Using the efficient DACS, the malignant cancer cells (MCF 7) were isolated successfully by noninvasive methods from normal cells with similar cell size distributions (MCF 10A), depending on differences between their material properties such as conductivity and permittivity, because our system was able to discern the subtle differences in the properties by generating continuously changed electrical field gradients. In order to evaluate the separation performance without considering size variations, the cells collected from each outlet were divided into size-dependent groups and counted statistically. Following that, the quantitative relative ratio of numbers between MCF 7 and MCF 10A cells in each size-dependent group separated by the DEP were compared according to applied frequencies in the range 48, 51, and 53 MHz with an applied amplitude of 8 Vpp. Finally, under the applied voltage of 48 MHz–8 Vpp and a flow rate of 290 μm/s, MCF 7 and MCF 10A cells were separated with a maximum efficiency of 86.67% and 98.73% respectively. Therefore, our suggested system shows it can be used for detection and separation of cancerous epithelial cells from noncancerous cells in clinical applications. [ABSTRACT FROM AUTHOR]

Digital microfluidics has recently emerged as a new paradigm in the world of lab-on-a-chip technology. A wide variety of bioanalyses have been successfully implemented in this format. This paper reviews the various techniques that have been adapted to digital microfluidic systems, and the current state of the field. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal d-Ala- d-Ala intermediates in the peptidoglycan of the cell wall of Gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two d-Ala- d-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled d-Ala- d-Ala- d-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 × 10−6 and 3.0 × 10−6 mol L−1, respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of Gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide–antibiotic interaction. [ABSTRACT FROM AUTHOR]

The paper reviews the state-of-art for micro optical fluidic systems (MOFS), or optofluidics, which employs optics and fluidics in a microsystem environment to perform novel functionalities and in-depth analysis in the biophysical area. Various topics, which include the introduction of MOFS in biomedical engineering, the implementation of near-field optics and also the applications of MOFS to biophysical studies, are discussed. Different optical detection techniques, such as evanescent wave, surface plasmon resonance, surface enhanced Raman scattering, resonators and transistors, have been studied extensively and integrated into MOFS. In addition, MOFS also provides a platform for various studies of cell biophysics, such as cell mass determination and cell Young’s modulus measurement. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

This paper describes an innovative integrated micro flow cytometer that presents a new arrangement for the excitation/detection system. The sample liquid, containing the fluorescent marked particles/cells under analysis, is hydrodynamically squeezed into a narrow stream by two sheath flows so that the particles/cells flow individually through a detection region. The detection of the particles/cells emitted fluorescence is carried out by using a collection fiber placed orthogonally to the flow. The device is based on silicon hollow core antiresonant reflecting optical waveguides (ARROWs). ARROW geometry allows one to use the same channel to guide both the sample stream and the fluorescence excitation light, leading to a simplification of the optical configuration and to an increase of the signal-to-noise ratio. The integrated micro flow cytometer has been characterized by using biological samples marked with standard fluorochromes. The experimental investigation confirms the success of the proposed microdevice in the detection of cells. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

This paper reviews applications of laser-based techniques to the fabrication of microfluidic devices for biochips and addresses some of the challenges associated with the manufacture of these devices. Special emphasis is placed on the use of lasers for the rapid prototyping and production of biochips in particular for applications in which silicon is not the preferred material base. Part I of this review addresses applications and devices using UV lasers for laser ablation and surface treatment of microchannels, in particular in polymers. [ABSTRACT FROM AUTHOR]

This paper presents a new approach for the metrological characterization of microfabricated features on microfluidic chips, based on a combination of poly(dimethylsiloxane) (PDMS) replication and charge-coupled device (CCD) imaging. A PDMS replicate was cast from the original chip sample, and a 2-mm thick sample slice was cut from the replica at the cross-section to be studied. The digital image of the revealed structural profile was captured by a CCD camera under a microscope, and the image was processed using specially-developed algorithms for CCD image calibration and edge detection. Depth and width measurements obtained using the method agreed well with those gained using a stylus profiler and universal measuring microscope, with a deviation of below 0.9 µm, while profile distortions of deeper structures using stylus profilers were avoided. The method is reliable, non-destructive, and cheap and simple to implement in any analytical laboratory. [ABSTRACT FROM AUTHOR]

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome–target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol μL-1 (10 pmol L-1) . Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system. [ABSTRACT FROM AUTHOR]

To tackle cancer and provide prompt diagnoses and prognoses, the constantly evolving biosensing field is continuously on the lookout for novel markers that can be non-invasively analysed. Extracellular vesicles (EVs) may represent a promising biomarker that also works as a source of biomarkers. The augmented cellular activity of cancerous cells leads to the production of higher numbers of EVs, which can give direct information on the disease due to the presence of general and cancer-specific surface-tethered molecules. Moreover, the intravesicular space is enriched with other molecules that can considerably help in the early detection of neoplasia. Even though EV-targeted research has indubitably received broad attention lately, there still is a wide lack of practical and effective quantitative procedures due to difficulties in pre-analytical and analytical phases. This review aims at providing an exhaustive outline of the recent progress in EV detection using electrochemical and photoelectrochemical biosensors, with a focus on handling approaches and trends in the selection of bioreceptors and molecular targets related to EVs that might guide researchers that are approaching such an unstandardised field. [ABSTRACT FROM AUTHOR]