We present a new, power-free and flexible detection system named MCFphone for portable colorimetric and fluorescence quantitative sandwich immunoassay detection of prostate specific antigen (PSA). The MCFphone is composed by a smartphone integrated with a magnifying lens, a simple light source and a miniaturised immunoassay platform, the Microcapillary Film (MCF). The excellent transparency and flat geometry of fluoropolymer MCF allowed quantitation of PSA in the range 0.9 to 60 ng/ml with<7% precision in 13 min using enzymatic amplification and a chromogenic substrate. The lower limit of detection was further improved from 0.4 to 0.08 ng/ml in whole blood samples with the use of a fluorescence substrate. The MCFphone has shown capable of performing rapid (13 to 22 min total assay time) colorimetric quantitative and highly sensitive fluorescence tests with good %Recovery, which represents a major step in the integration of a new generation of inexpensive and portable microfluidic devices with commercial immunoassay reagents and off-the-shelf smartphone technology.
We present a new concept for rapid and fully portable prostate specific antigen (PSA) measurements, termed "lab-in-a-briefcase", which integrates an affordable microfluidic ELISA platform utilising a melt-extruded fluoropolymer microcapillary film (MCF) containing an array of 10 200 μm internal diameter capillaries, a disposable multi-syringe aspirator (MSA), a sample tray pre-loaded with all of the required immunoassay reagents, and a portable film scanner for colorimetric signal digital quantification. Each MSA can perform 10 replicate microfluidic immunoassays on 8 samples, allowing 80 measurements to be made in less than 15 minutes based on semi-automated operation, without the need of additional fluid handling equipment. The assay was optimised for the measurement of a clinically relevant range of PSA of 0.9 to 60.0 ng ml(-1) in 15 minutes with CVs on the order of 5% based on intra-assay variability when read using a consumer flatbed film scanner. The PSA assay performance in the MSA remained robust in undiluted or 1 : 2 diluted human serum or whole blood, and the matrix effect could simply be overcome by extending sample incubation times. The PSA "lab-in-a-briefcase" is particularly suited to a low-resource health setting, where diagnostic labs and automated immunoassay systems are not accessible, by allowing PSA measurement outside the laboratory using affordable equipment.
Biosensors devices have attracted the attention of many researchers across the world. They have the capability to solve a large number of analytical problems and challenges. They are future ubiquitous devices for disease diagnosis, monitoring, treatment and health management. This review presents an overview of the biosensors field, highlighting the current research and development of bio-integrated and implanted biosensors. These devices are micro- and nano-fabricated, according to numerous techniques that are adapted in order to offer a suitable mechanical match of the biosensor to the surrounding tissue, and therefore decrease the body’s biological response. For this, most of the skin-integrated and implanted biosensors use a polymer layer as a versatile and flexible structural support, combined with a functional/active material, to generate, transmit and process the obtained signal. A few challenging issues of implantable biosensor devices, as well as strategies to overcome them, are also discussed in this review, including biological response, power supply, and data communication.
The latest clinical procedures for the timely and cost-effective diagnosis of chronic and acute clinical conditions, such as cardiovascular diseases, cancer, chronic respiratory diseases, diabetes or sepsis (i.e. the biggest causes of death worldwide), involve the quantitation of specific protein biomarkers released into the blood stream or other physiological fluids (e.g. urine or saliva). The clinical thresholds are usually in the femtomolar to picolomar range, and consequently the measurement of these protein biomarkers heavily relies on highly sophisticated, bulky and automated equipment in centralised pathology laboratories. The first microfluidic devices capable of measuring protein biomarkers in miniaturised immunoassays were presented nearly two decades ago and promised to revolutionise point-of-care (POC) testing by offering unmatched sensitivity and automation in a compact POC format; however, the development and adoption of microfluidic protein biomarker tests has fallen behind expectations. This review presents a detailed critical overview into the pipeline of microfluidic devices developed in the period 2005-2016 capable of measuring protein biomarkers from the pM to fM range in formats compatible with POC testing, with a particular focus on the use of affordable microfluidic materials and compact low-cost signal interrogation. The integration of these two important features (essential unique selling points for the successful microfluidic diagnostic products) has been missed in previous review articles and explain the poor adoption of microfluidic technologies in this field. Most current miniaturised devices compromise either on the affordability, compactness and/or performance of the test, making current tests unsuitable for the POC measurement of protein biomarkers. Seven core technical areas, including (i) the selected strategy for antibody immobilisation, (ii) the surface area and surface-area-to-volume ratio, (iii) surface passivation, (iv) the biological matrix interference, (v) fluid control, (vi) the signal detection modes and (vii) the affordability of the manufacturing process and detection system, were identified as the key to the effective development of a sensitive and affordable microfluidic protein biomarker POC test.
Sensitive quantitation of multiple cytokines can provide important diagnostic information during infection, inflammation and immunopathology. In this study sensitive immunoassay detection of human cytokines IL-1β, IL-6, IL-12p70 and TNFα is shown for singleplex and multiplex formats using a novel miniaturized ELISA platform. The platform uses a disposable plastic multi-syringe aspirator (MSA) integrating 8 disposable fluoropolymer microfluidic test strips, each containing an array of ten 200 μm mean i.d. microcapillaries coated with a set of monoclonal antibodies. Each MSA device thus performs 10 tests on 8 samples, delivering 80 measurements. Unprecedented levels of sensitivity were obtained with the novel fluoropolymer microfluidic material and simple colorimetric detection in a flatbed scanner. The limit of detection for singleplex detection ranged from 2.0 to 15.0 pg ml(-1), i.e. 35 and 713 femtomolar for singleplex cytokine detection, and the intra- and inter-assay coefficient of variation (CV) remained within 10%. In addition, a triplex immunoassay was developed for measuring IL-1β, IL-12p70 and TNFα simultaneously from a given sample in the pg ml(-1) range. These assays permit high sensitivity measurement with rapid <15 min assay or detection from undiluted blood serum. The portability, speed and low-cost of this system are highly suited to point-of-care testing and field diagnostics applications.
Cardiovascular diseases, cancer, and diabetes are high mortality diseases, which account for almost two thirds of all deaths worldwide. Their early detection and continuous evaluation are fundamental for an improved patient prognosis and reduced socioeconomic impact. Current biosensor technologies are typically based on the analysis of whole blood samples from patients for the detection of disease-specific biomarkers. However, these technologies display serious shortcomings, such as reduced sensitivity and dynamic range, limited in vivo applicability, and lack of continuous monitoring. There is the urgent need for new diagnostic and treatment follow-up tools, which allow for the early detection of the pathology as well as for the continuous monitoring of the physiological responses to specific therapies. During the last years, a new generation of biosensor technologies with improved performance has emerged in the biomedical sector. The combination of advanced biomaterial methods, biochemical tools, and micro/nanotechnology approaches has resulted in the development of innovative three-dimensional (3D) biosensor platforms for advanced medical diagnosis. In this review, we report the most recent advances in the field of 3D biosensors for clinical applications, focusing on the diagnosis and monitoring of cardiovascular diseases, cancer, and diabetes. We discuss about their clinical performance compared to standard biosensor technologies, their implantable capability, and their integration into microfluidic devices to develop clinically-relevant models. Overall, we anticipate that 3D biosensors will drive us toward a new paradigm in medical diagnosis, resulting in real-time in vivo biosensors capable to significantly improve patient prognosis.
This study reports for the first time the sensitive colorimetric and fluorescence detection of clinically relevant protein biomarkers by sandwich immunoassays using the covalent immobilisation of antibodies onto the fluoropolymer surface inside Teflon®-FEP microfluidic devices. Teflon®-FEP has outstanding optical transparency ideal for high-sensitivity colorimetric and fluorescence bioassays, however this thermoplastic is regarded as chemically inert and very hydrophobic. Covalent immobilisation can offer benefits over passive adsorption to plastic surfaces by allowing better control over antibody density, orientation and analyte binding capacity, and so we tested a range of different and novel covalent immobilisation strategies. We first functionalised the inner surface of a 10-bore, 200 μm internal diameter FEP microcapillary film with high-molecular weight polyvinyl alcohol (PVOH) without changing the outstanding optical transparency of the device delivered by the matched refractive index of FEP and water. Glutaraldehyde immobilisation was compared with the use of photoactivated linkers and NHS-ester crosslinkers for covalently immobilising capture antibodies onto PVOH. Three clinically relevant sandwich ELISAs were tested against the cytokine IL-1β, the myocardial infarct marker cardiac troponin I (cTnI), and the chronic heart failure marker brain natriuretic peptide (BNP). Overall, glutaraldehyde immobilisation was effective for BNP assays, but yielded unacceptable background for IL-1β and cTnI assays caused by direct binding of the biotinylated detection antibody to the modified PVOH surface. We found NHS-ester groups reacted with APTES-treated PVOH coated fluoropolymers. This facilitated a novel method for capture antibody immobilisation onto fluoropolymer devices using a bifunctional NHS-maleimide crosslinker. The density of covalently immobilised capture antibodies achieved using PVOH/APTES/NHS/maleimide approached levels seen with passive adsorption, and sensitive and quantitative assay performance was achieved using this method. Overall, the PVOH coating provided an excellent surface for controlled covalent antibody immobilisation onto Teflon®-FEP for performing high-sensitivity immunoassays.
Rapid and quantitative prostate-specific antigen (PSA) biomarker detection would be beneficial to cancer diagnostics, improving early detection and therefore increasing chances of survival. Nanoparticle-based detection is routinely used in one-step nitrocellulose-based lateral flow (LF) immunoassays; however, it is well established within the scientific diagnostic community that LF technology lacks sensitivity for measuring biomarkers, such as prostate-specific antigen (PSA). A trend in point-of-care (POC) protein biomarker quantitation is the miniaturization of immunoassays in microfluidic devices. This work aimed at testing the feasibility of carbon and gold nanoparticles as immunoassay labels for PSA detection with cost-effective optical detection in a novel microfluidic POC platform called microcapillary film (MCF), consisting of a parallel array of fluoropolymer microcapillaries with 200-μm internal diameter. With neutravidin-coated carbon, nanoparticles were able to quantify an immobilized biotinylated monoclonal antibody (coating solution from 10 to 40 μg/ml) and PSA was successfully quantified in a sandwich assay using silver-enhanced gold nanoparticles and a flatbed scanner; yet, the dynamic range was limited to 10–100 ng/ml. Although direct optical detection of PSA without enzymatic amplification or fluorophores is possible and technically appealing for the simplified fluidics and signal scanning setups involved, ultimately, the binding of a thin layer of nanoparticles onto the wall of transparent microcapillaries is not sufficient to cause a significant drop on the optical colorimetric signal. Future studies will explore the use of fluorescence nanoparticles.
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