A Systematic Study and Potential Limitations of Proton-ELISA Platform for α-Synuclein Antigen Detection

To evaluate point-of-care testing (POCT) for the potential early detection of biomarkers of Parkinson’s disease, a systematic investigation of portable and low-cost platforms is performed based on the Proton-enzyme-linked immunosorbent assay (Proton-ELISA) methodology. The detection of the α-synuclein antigen was first presented by biotin-relative linkers, and glucose substrate solution was first performed with a systematic experimental design to optimize the sensing results. All materials in this study are commercially available. Three different experiments with the partitional check were performed to investigate the Proton-ELISA platform, including proton catalyzed efficiency, blocking efficiency, and full Proton-ELISA procedure. The response time was selected as 15 min by the time-dependent curves of a full reaction. The limit of detection of conventional ELISA kits is 0.169 ng/mL, which is much lower than the Proton-ELISA results. The final response of the full Proton-ELISA procedure to pH changes was approximately 0.60 and 0.12 for α-synuclein antigen concentrations of 100 ng/mL and 4 ng/mL, respectively. With the partitional check, pH changes of pure glucose substrate and conjugated oxidase and interference of the nonspecific binding are 1.7 and 0.04, respectively. The lower pH changes far from the partitional check results can be concluded for the properties of glucose oxidase conjugation, including the isoelectric point and binding affinity modification by the testing environment. This preliminary guideline can be used as a lesson learnt to speed up following studies of the evaluation and optimization of other antigen detection. Therefore, Proton-ELISA can be suggested for some special applications with the help of custom-designed conjugation in the environment with less degradation or interference and a proper detection concentration range.

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Colorimetric Diagnostic Capillary Enabled by Size Sieving in a Porous Hydrogel

Biosensors ◽

10.3390/bios10100130 ◽

2020 ◽

Vol 10 (10) ◽

pp. 130

Author(s):

John Mello Camille C. Guzman ◽

Sheng-Min Hsu ◽

Han-Sheng Chuang

Keyword(s):

Limit Of Detection ◽

Capillary Tube ◽

Point Of Care ◽

Low Cost ◽

Glass Capillary ◽

Diagnostic Device ◽

Early Screening ◽

Resource Limited ◽

Detection Of Biomarkers ◽

Porous Hydrogel

Handy and disposable point-of-care diagnostics facilitate the early screening of severe diseases in resource-limited areas. To address urgent needs in inconvenient sites, a simple colorimetric diagnostic device equipped with a capillary tube with porous hydrogel and immunocomplex particles was developed for the rapid detection of biomarkers (16 min). In this device, probe particles attach to capture particles (dp = 40 µm) and form sandwiched immunocomplexes in the presence of target biomarkers, and a red color progressively emerges when the sandwiched immunocomplex particles are blocked by the porous hydrogel embedded inside the glass capillary. Colorimetric aggregation was recorded using a smartphone and analyzed with imaging software. The limit of detection reached 1 ng/mL and showed a maximum of 79% accuracy compared with that obtained through a conventional spectrophotometric technique. The level of a diabetic retinopathy (DR) biomarker, lipocalin-1 (LCN-1), was measured in 1 µL of a human tear sample and used in testing the practicability of the proposed device. All healthy subjects showed lower intensity levels than the other diabetic counterparts (proliferative DR or nonproliferative DR patients), implying the potential of this device in clinical applications. Overall, the diagnostic device facilitates point-of-care-testing and provides a low-cost (~1 USD), compact, and reliable tool for early diagnosis in resource-limited areas.

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

Sensors ◽

10.3390/s21123985 ◽

2021 ◽

Vol 21 (12) ◽

pp. 3985

Author(s):

Nan Wan ◽

Yu Jiang ◽

Jiamei Huang ◽

Rania Oueslati ◽

Shigetoshi Eda ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Clinical Diagnostics ◽

Detection Methods ◽

Capacitive Sensing ◽

Application Potential ◽

Mirna Detection ◽

Mirna 25 ◽

Low Sensitivity

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

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Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein

Biosensors ◽

10.3390/bios11010004 ◽

2020 ◽

Vol 11 (1) ◽

pp. 4

Author(s):

Donggee Rho ◽

Seunghyun Kim

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Optical Cavity ◽

Sample Volume ◽

Small Sample ◽

Label Free ◽

C Reactive Protein ◽

Reactive Protein ◽

Cavity Structure

An optical cavity-based biosensor (OCB) has been developed for point-of-care (POC) applications. This label-free biosensor employs low-cost components and simple fabrication processes to lower the overall cost while achieving high sensitivity using a differential detection method. To experimentally demonstrate its limit of detection (LOD), we conducted biosensing experiments with streptavidin and C-reactive protein (CRP). The optical cavity structure was optimized further for better sensitivity and easier fluid control. We utilized the polymer swelling property to fine-tune the optical cavity width, which significantly improved the success rate to produce measurable samples. Four different concentrations of streptavidin were tested in triplicate, and the LOD of the OCB was determined to be 1.35 nM. The OCB also successfully detected three different concentrations of human CRP using biotinylated CRP antibody. The LOD for CRP detection was 377 pM. All measurements were done using a small sample volume of 15 µL within 30 min. By reducing the sensing area, improving the functionalization and passivation processes, and increasing the sample volume, the LOD of the OCB are estimated to be reduced further to the femto-molar range. Overall, the demonstrated capability of the OCB in the present work shows great potential to be used as a promising POC biosensor.

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Whole Blood Immunoassay Based on Centrifugal Bead Sedimentation

Clinical Chemistry ◽

10.1373/clinchem.2011.162206 ◽

2011 ◽

Vol 57 (5) ◽

pp. 753-761 ◽

Cited By ~ 44

Author(s):

Ulrich Y Schaff ◽

Greg J Sommer

Keyword(s):

Whole Blood ◽

Single Channel ◽

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Microfluidic System ◽

Blood Samples ◽

Reactive Protein ◽

Immunoassay Technique ◽

Microfluidic Immunoassay

BACKGROUND Centrifugal “lab on a disk” microfluidics is a promising avenue for developing portable, low-cost, automated immunoassays. However, the necessity of incorporating multiple wash steps results in complicated designs that increase the time and sample/reagent volumes needed to run assays and raises the probability of errors. We present proof of principle for a disk-based microfluidic immunoassay technique that processes blood samples without conventional wash steps. METHODS Microfluidic disks were fabricated from layers of patterned, double-sided tape and polymer sheets. Sample was mixed on-disk with assay capture beads and labeling antibodies. Following incubation, the assay beads were physically separated from the blood cells, plasma, and unbound label by centrifugation through a density medium. A signal-laden pellet formed at the periphery of the disk was analyzed to quantify concentration of the target analyte. RESULTS To demonstrate this technique, the inflammation biomarkers C-reactive protein and interleukin-6 were measured from spiked mouse plasma and human whole blood samples. On-disk processing (mixing, labeling, and separation) facilitated direct assays on 1-μL samples with a 15-min sample-to-answer time, <100 pmol/L limit of detection, and 10% CV. We also used a unique single-channel multiplexing technique based on the sedimentation rate of different size or density bead populations. CONCLUSIONS This portable microfluidic system is a promising method for rapid, inexpensive, and automated detection of multiple analytes directly from a drop of blood in a point-of-care setting.

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Two Potential Clinical Applications of Origami-Based Paper Devices

Diagnostics ◽

10.3390/diagnostics9040203 ◽

2019 ◽

Vol 9 (4) ◽

pp. 203

Author(s):

Zong-Keng Kuo ◽

Tsui-Hsuan Chang ◽

Yu-Shin Chen ◽

Chao-Min Cheng ◽

Chia-Ying Tsai

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Test Sample ◽

Clinical Applications ◽

Ease Of Use ◽

Enzyme Linked Immunosorbent Assay ◽

Elisa System ◽

Test Zone ◽

Moist Environment ◽

Simplex Virus

Detecting small amounts of analyte in clinical practice is challenging because of deficiencies in specimen sample availability and unsuitable sampling environments that prevent reliable sampling. Paper-based analytical devices (PADs) have successfully been used to detect ultralow amounts of analyte, and origami-based PADs (O-PADs) offer advantages that may boost the overall potential of PADs in general. In this study, we investigated two potential clinical applications for O-PADs. The first O-PAD we investigated was an origami-based enzyme-linked immunosorbent assay (ELISA) system designed to detect different concentrations of rabbit IgG. This device was designed with four wing structures, each of which acted as a reagent loading zone for pre-loading ELISA reagents, and a central test sample loading zone. Because this device has a low limit of detection (LOD), it may be suitable for detecting IgG levels in tears from patients with a suspected viral infection (such as herpes simplex virus (HSV)). The second O-PAD we investigated was designed to detect paraquat levels to determine potential poisoning. To use this device, we sequentially folded each of two separate reagent zones, one preloaded with NaOH and one preloaded with ascorbic acid (AA), over the central test zone, and added 8 µL of sample that then flowed through each reagent zone and onto the central test zone. The device was then unfolded to read the results on the test zone. The three folded layers of paper provided a moist environment not achievable with conventional paper-based ELISA. Both O-PADs were convenient to use because reagents were preloaded, and results could be observed and analyzed with image analysis software. O-PADs expand the testing capacity of simpler PADs while leveraging their characteristic advantages of convenience, cost, and ease of use, particularly for point-of-care diagnosis.

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Recent Developments of Electrochemical and Optical Biosensors for Antibody Detection

International Journal of Molecular Sciences ◽

10.3390/ijms21010134 ◽

2019 ◽

Vol 21 (1) ◽

pp. 134 ◽

Cited By ~ 5

Author(s):

Wei Xu ◽

Daniel Wang ◽

Derek Li ◽

Chung Chiun Liu

Keyword(s):

Point Of Care ◽

Low Cost ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Disease Diagnosis ◽

Antibody Detection ◽

Recent Developments ◽

Care Systems ◽

Point Of Care Systems ◽

Detection Of Biomarkers

Detection of biomarkers has raised much interest recently due to the need for disease diagnosis and personalized medicine in future point-of-care systems. Among various biomarkers, antibodies are an important type of detection target due to their potential for indicating disease progression stage and the efficiency of therapeutic antibody drug treatment. In this review, electrochemical and optical detection of antibodies are discussed. Specifically, creating a non-label and reagent-free sensing platform and construction of an anti-fouling electrochemical surface for electrochemical detection are suggested. For optical transduction, a rapid and programmable platform for antibody detection using a DNA-based beacon is suggested as well as the use of bioluminescence resonance energy transfer (BRET) switch for low cost antibody detection. These sensing strategies have demonstrated their potential for resolving current challenges in antibody detection such as high selectivity, low operation cost, simple detection procedures, rapid detection, and low-fouling detection. This review provides a general update for recent developments in antibody detection strategies and potential solutions for future clinical point-of-care systems.

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Application of Flexible Display Technology to Low Cost Multiplexed Point-of-Care Medical Diagnostic Testing

Journal of Global Oncology ◽

10.1200/jgo.2016.003855 ◽

2016 ◽

Vol 2 (3_suppl) ◽

pp. 14s-14s

Author(s):

Benjamin A. Katchman ◽

Joseph T. Smith ◽

Jennifer Blain Christen ◽

Karen S. Anderson

Keyword(s):

Intellectual Property ◽

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Manufacturing Technology ◽

Analytical Sensitivity ◽

State University ◽

Arizona State University ◽

New Approach ◽

Display Technology

Abstract 62 One of the key roadblocks limiting the transition of high-sensitivity and high-specificity point-of-care technologies from the research laboratory to wide spread use is the availability of a low-cost-high-volume manufacturing technology. This work presents a new interdisciplinary approach combining low cost commercial display manufacturing technology with programmable high density protein microarray printing technology to fabricate disposable point-of-care immunosensors with clinical level sensitivity. Our approach is designed to leverage advances in commercial display technology to reduce pre-functionalized biosensor substrate costs to pennies per cm2, as well as to leverage the display industry’s ability to manufacture an immense number of low cost consumer electronic products annually. For this work, we demonstrate that our new approach can offer diagnostic sensitivity at or below 10 pg/mL, which approaches the lower limit of detection of typical clinical laboratory instrumentation. Our new approach is also designed to overcome the limited analytical sensitivity of existing POC devices (>100x improved sensitivity). It also contains new capability for multiplexed biomarker detection (>10 antigens) in a single low cost POC device through an innovative disposable and scalable architecture, based on flat panel display technology. Here, we demonstrate multiplexed detection of antibodies to the HPV16 proteins E2, E6, and E7, which are circulating biomarkers for cervical as well as head and neck cancers. This detection technology has 100 percent correlation to our current laboratory-based measurement instrumentation. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST: Benjamin A. Katchman Patents, Royalties, Other Intellectual Property: Arizona State University Joseph T. Smith Patents, Royalties, Other Intellectual Property: Arizona State University Jennifer Blain Christen Patents, Royalties, Other Intellectual Property: Arizona State University Karen S. Anderson Stock or Other Ownership: Provista Diagnostics Consulting or Advisory Role: Provista Diagnostics Patents, Royalties, Other Intellectual Property: Arizona State University

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Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform

Applied and Environmental Microbiology ◽

10.1128/aem.02449-16 ◽

2016 ◽

Vol 83 (4) ◽

Cited By ~ 25

Author(s):

Lars D. Renner ◽

Jindong Zan ◽

Linda I. Hu ◽

Manuel Martinez ◽

Pedro J. Resto ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Bacterial Pathogens ◽

Diagnostic System ◽

Molecular Diagnostic ◽

Technology Gap ◽

Content Type ◽

Antimicrobial Drug Resistance ◽

Industrial Processing

ABSTRACT An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.

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Inkjet printing of paraffin on paper allows low-cost point-of-care diagnostics for pathogenic fungi

Cellulose ◽

10.1007/s10570-020-03314-3 ◽

2020 ◽

Vol 27 (13) ◽

pp. 7691-7701 ◽

Cited By ~ 1

Author(s):

Anusha Prabhu ◽

M. S. Giri Nandagopal ◽

Prakash Peralam Yegneswaran ◽

Hardik Ramesh Singhal ◽

Naresh Kumar Mani

Keyword(s):

Filter Paper ◽

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Pathogenic Fungi ◽

Sem Analysis ◽

Qualitative Assessment ◽

Resource Limited ◽

Hydrophobic Barrier ◽

Pore Blockage

Abstract We present a high resolution, ultra-frugal printing of paper microfluidic devices using in-house paraffin formulation on a simple filter paper. The patterns printed using an office inkjet printer formed a selective hydrophobic barrier of 4 ± 1 µm thickness with a hydrophilic channel width of 275 µm. These printed patterns effectively confine common aqueous solutions and solvents, which was verified by solvent compatibility studies. SEM analysis reveals that the solvent confinement is due to pore blockage in the filter paper. The fabricated paper-based device was validated for qualitative assessment of Candida albicans (pathogenic fungi) by using a combination of L-proline β-naphthylamide as the substrate and cinnamaldehyde as an indicator. Our studies reveal that the pathogenic fungi can be detected within 10 min with the limit of detection (LOD) of 0.86 × 106 cfu/mL. Owing to its simplicity, this facile method shows high potential and can be scaled up for developing robust paper-based devices for biomarker detection in resource-limited settings. Graphic abstract

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Comparison of MagPix Assays and Enzyme-Linked Immunosorbent Assay for Detection of Hemorrhagic Fever Viruses

Journal of Clinical Microbiology ◽

10.1128/jcm.01693-16 ◽

2016 ◽

Vol 55 (1) ◽

pp. 68-78 ◽

Cited By ~ 21

Author(s):

Neal G. Satterly ◽

Matthew A. Voorhees ◽

Abbe D. Ames ◽

Randal J. Schoepp

Keyword(s):

Immunosorbent Assay ◽

Limit Of Detection ◽

Hemorrhagic Fever ◽

Antigen Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Hemorrhagic Fevers ◽

Viral Hemorrhagic Fevers ◽

Medical Countermeasures ◽

Potential Use ◽

Excellent Reproducibility

ABSTRACT Viral hemorrhagic fevers, because of their high mortality rates, the lack of medical countermeasures, and their potential use as instruments of bioterrorism, pose a significant threat to the developed and the developing areas of the world. The key to preventing the spread of these diseases is early and accurate detection. For decades, the gold-standard immunoassay for hemorrhagic fever detection has been the enzyme-linked immunosorbent assay (ELISA); however, newer technologies are emerging with increased sensitivities. One such technology is the Luminex MagPix platform using xMAP microspheres. Here, we compare the MagPix platform with a traditional ELISA for IgM and antigen detection of infections from Lassa and Ebola viruses (LASV and EBOV, respectively). For IgM detection in nonhuman primate samples, the MagPix platform was 5 and 25 times more sensitive in detecting LASV and EBOV, respectively, compared to that with ELISA. For antigen detection in buffer, the MagPix platform was 25 and 2.5 times more sensitive in detecting lower levels of LASV and EBOV, respectively. In both IgM and antigen detection assays, the MagPix platform demonstrated excellent reproducibility at the lower limit of detection (LLOD). These findings demonstrate that the MagPix platform is a viable diagnostic replacement for the ELISA for viral hemorrhagic fevers.

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