Detector-Free Photothermal Bar-Chart Microfluidic Chips (PT-Chips) for Visual Quantitative Detection of Biomarkers

The volumetric bar-chart microfluidic chips (V-Chips) driven by chemical reaction-generated gas provide a promising platform for point-of-care (POC) visual biomarker quantitation. However, multiple limitations are encountered in conventional V-Chips, such as costly and complex chip fabrication, complicated assembly, and imprecise controllability of gas production. Herein, we introduced nanomaterial-mediated photothermal effects to V-Chips, and for the first time developed a new type of V-Chip, photothermal bar-chart microfluidic chip (PT-Chip), for visual quantitative detection of biochemicals without any bulky and costly analytical instruments. Immunosensing signals were converted to visual readout signals via photothermal effects, the on-chip bar-chart movements, enabling quantitative biomarker detection on a low-cost polymer hybrid PT-Chip with on-chip scale rulers. Four different human serum samples containing prostate-specific antigen (PSA) as a model analyte were detected simultaneously using the PT-Chip, with the limit of detection of 2.1 ng/mL, meeting clinical diagnostic requirements. Although no conventional signal detectors were used, it achieved comparable detection sensitivity to absorbance measurements with a microplate reader. The PT-Chip was further validated by testing human whole blood without the color interference problem, demonstrating good analytical performance of our method even in complex matrixes and thus the potential to fill a gap in current clinical diagnostics that is incapable of testing whole blood. This new PT-Chip driven by nanomaterial-mediated photothermal effects opens a new horizon of microfluidic platforms for instrument-free diagnostics at the point of care.

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Detector-Free Photothermal Bar-Chart Microfluidic Chips (PT-Chips) for Visual Quantitative Detection of Biomarkers

10.26434/chemrxiv.14131514 ◽

2021 ◽

Author(s):

Wan Zhou ◽

Guanglei Fu [email protected] ◽

Xiujun Li

Keyword(s):

Whole Blood ◽

Limit Of Detection ◽

Point Of Care ◽

Clinical Diagnostics ◽

Detection Sensitivity ◽

Quantitative Detection ◽

Microfluidic Chips ◽

Analytical Instruments ◽

Photothermal Effects ◽

On Chip

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PERFORMANCE CHARACTERISTICS OF A FRET-BASED IMMUNOASSAY FOR QUANTITATION OF INFLIXIMAB ON A POINT-OF-CARE INSTRUMENT SYSTEM

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.135 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S57-S57

Author(s):

Edgar Ong ◽

Ruo Huang ◽

Richard Kirkland ◽

Michael Hale ◽

Larry Mimms

Keyword(s):

Whole Blood ◽

Limit Of Detection ◽

Point Of Care ◽

Quantitative Detection ◽

Therapeutic Drug ◽

Analytical Sensitivity ◽

Sample Type ◽

Hook Effect ◽

Limit Of Quantitation ◽

Assay Precision

Abstract Introduction A fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of infliximab (IFX) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise IFX assay and ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise IFX assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of IFX into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of IFX. Linearity was determined by testing IFX across the assay range. Hook effect was assessed at IFX concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise IFX assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER infliximab ELISA test (Theradiag, France). The accuracy of the Procise IFX assay is established through calibrators and controls traceable to the WHO 1st International Standard for Infliximab (NIBSC code: 16/170). Results The Procise IFX assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 1.1 µg/mL in serum and 0.6, 1.1, and 1.7 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.7 to 77.2 µg/mL in serum and whole blood. No hook effect was observed at an IFX concentration of 200 µg/mL as the value reported as “>ULoQ”. Assay precision testing across 20 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.7%, an inter-assay CV of <2%, and a total CV of 3.4%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±8% of control. Testing of biosimilars (infliximab-dyyb and infliximab-abda) showed good recovery. A good correlation to the Theradiag infliximab ELISA was obtained for both serum (slope=1.01; r=0.99) and whole blood (slope=1.01; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise IFX assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows very good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise IFX assay ideal for obtaining fast and accurate IFX quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

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PERFORMANCE CHARACTERISTICS OF A FRET-BASED IMMUNOASSAY FOR QUANTITATION OF ADALIMUMAB ON A POINT-OF-CARE INSTRUMENT SYSTEM

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.134 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S56-S57

Author(s):

Edgar Ong ◽

Ruo Huang ◽

Richard Kirkland ◽

Michael Hale ◽

Larry Mimms

Keyword(s):

Whole Blood ◽

Limit Of Detection ◽

Point Of Care ◽

Quantitative Detection ◽

Therapeutic Drug ◽

Analytical Sensitivity ◽

Sample Type ◽

Hook Effect ◽

Limit Of Quantitation ◽

Assay Precision

Abstract Introduction A fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of adalimumab (ADL) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise ADL assay and the ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise ADL assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of ADL into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of ADL. Linearity was determined by testing ADL across the assay range. Hook effect was assessed at ADL concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise ADL assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER adalimumab ELISA test (Theradiag, France). The accuracy of the Procise ADL assay is established through calibrators and controls traceable to the WHO 1st International Standard for Adalimumab (NIBSC code: 17/236). Results The Procise ADL assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 0.6 µg/mL in serum and 0.5, 0.9, and 1.3 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.3 to 51.5 µg/mL in serum and whole blood. No hook effect was observed at an ADL concentration of 200 µg/mL as the value reported as “>ULoQ”. Assay precision testing across 10 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.8%, an inter-assay CV of ≤1.5%, and a total CV of 3.5%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±7.4% of control. Testing of biosimilars (adalimumab-atto and adalimumab-xxxx) showed good recovery. A good correlation to the Theradiag adalimumab ELISA was obtained for both serum (slope=0.94; r=0.99) and whole blood (slope=1.13; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise ADL assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise ADL assay ideal for obtaining fast and accurate ADL quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

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Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

10.26434/chemrxiv.5662315.v1 ◽

2017 ◽

Author(s):

Bo Tian ◽

Peter Svedlindh ◽

Mattias Strömberg ◽

Erik Wetterskog

Keyword(s):

Magnetic Nanoparticles ◽

Ferromagnetic Resonance ◽

Limit Of Detection ◽

Point Of Care ◽

Rolling Circle Amplification ◽

Resonance Field ◽

Isothermal Amplification ◽

Detection Sensitivity ◽

Serum Samples ◽

Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, i.e., rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg2P2O7 (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.

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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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Monolithic integration of nanorod arrays on microfluidic chips for fast and sensitive one-step immunoassays

Microsystems & Nanoengineering ◽

10.1038/s41378-021-00291-w ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Ye Wang ◽

Jiongdong Zhao ◽

Yu Zhu ◽

Shurong Dong ◽

Yang Liu ◽

...

Keyword(s):

Troponin I ◽

Specific Antigen ◽

Monolithic Integration ◽

Detection Sensitivity ◽

Quantitative Detection ◽

Plasmonic Nanostructures ◽

Nanorod Arrays ◽

Microfluidic Chips ◽

One Step ◽

Flow Through

AbstractHere, we present integrated nanorod arrays on microfluidic chips for fast and sensitive flow-through immunoassays of physiologically relevant macromolecules. Dense arrays of Au nanorods are easily fabricated through one-step oblique angle deposition, which eliminates the requirement of advanced lithography methods. We report the utility of this plasmonic structure to improve the detection limit of the cardiac troponin I (cTnI) assay by over 6 × 105-fold, reaching down to 33.9 fg mL−1 (~1.4 fM), compared with an identical assay on glass substrates. Through monolithic integration with microfluidic elements, the device enables a flow-through assay for quantitative detection of cTnI in the serum with a detection sensitivity of 6.9 pg mL−1 (~0.3 pM) in <6 min, which was 4000 times lower than conventional glass devices. This ultrasensitive detection arises from the large surface area for antibody conjugation and metal-enhanced fluorescent signals through plasmonic nanostructures. Moreover, due to the parallel arrangement of flow paths, simultaneous detection of multiple cancer biomarkers, including prostate-specific antigen and carcinoembryonic antigen, has been fulfilled with increased signal-to-background ratios. Given the high performance of this assay, together with its simple fabrication process that is compatible with standard mass manufacturing techniques, we expect that the prepared integrated nanorod device can bring on-site point-of-care diagnosis closer to reality.

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A Smartphone Camera Colorimetric Assay of Acetylcholinesterase and Butyrylcholinesterase Activity

Sensors ◽

10.3390/s21051796 ◽

2021 ◽

Vol 21 (5) ◽

pp. 1796

Author(s):

Miroslav Pohanka ◽

Jitka Zakova

Keyword(s):

Limit Of Detection ◽

Colorimetric Assay ◽

Point Of Care ◽

Specific Treatment ◽

Point Of Care Testing ◽

Analytical Instruments ◽

Colorimetric Test ◽

3D Printed ◽

Butyrylcholinesterase Activity ◽

Additional Education

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can serve as biochemical markers of various pathologies like liver disfunction and poisonings by nerve agents. Ellman’s assay is the standard spectrophotometric method to measure cholinesterase activity in clinical laboratories. The authors present a new colorimetric test to assess AChE and BChE activity in biological samples using chromogenic reagents, treated 3D-printed measuring pads and a smartphone camera as a signal detector. Multiwell pads treated with reagent substrates 2,6-dichlorophenolindophenyl acetate, indoxylacetate, ethoxyresorufin and methoxyresorufin were prepared and tested for AChE and BChE. In the experiments, 3D-printed pads containing indoxylacetate as a chromogenic substrate were optimal for analytical purposes. The best results were achieved using the red (R) channel, where the limit of detection was 4.05 µkat/mL for BChE and 4.38 µkat/mL for AChE using a 40 µL sample and a 60 min assay. The major advantage of this method is its overall simplicity, as samples are applied directly without any specific treatment or added reagents. The assay was also validated to the standard Ellman’s assay using human plasma samples. In conclusion, this smartphone camera-based colorimetric assay appears to have practical applicability and to be a suitable method for point-of-care testing because it does not require specific manipulation, additional education of staff or use of sophisticated analytical instruments.

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Development of a Pharmacogenetic Lab-on-Chip Assay Based on the In-Check Technology to Screen for Genetic Variations Associated to Adverse Drug Reactions to Common Chemotherapeutic Agents

Biosensors ◽

10.3390/bios10120202 ◽

2020 ◽

Vol 10 (12) ◽

pp. 202

Author(s):

Rosario Iemmolo ◽

Valentina La Cognata ◽

Giovanna Morello ◽

Maria Guarnaccia ◽

Mariamena Arbitrio ◽

...

Keyword(s):

Genetic Polymorphisms ◽

Adverse Drug Reactions ◽

Limit Of Detection ◽

Chemotherapeutic Agents ◽

Clinical Diagnostics ◽

Individual Risk ◽

Chip Assay ◽

Drug Reactions ◽

Lab On Chip ◽

On Chip

Background: Antineoplastic agents represent the most common class of drugs causing Adverse Drug Reactions (ADRs). Mutant alleles of genes coding for drug-metabolizing enzymes are the best studied individual risk factors for these ADRs. Although the correlation between genetic polymorphisms and ADRs is well-known, pharmacogenetic tests are limited to centralized laboratories with expensive or dedicated instrumentation used by specialized personnel. Nowadays, DNA chips have overcome the major limitations in terms of sensibility, specificity or small molecular detection, allowing the simultaneous detection of several genetic polymorphisms with time and costs-effective advantages. In this work, we describe the design of a novel silicon-based lab-on-chip assay able to perform low-density and high-resolution multi-assay analysis (amplification and hybridization reactions) on the In-Check platform. Methods: The novel lab-on-chip was used to screen 17 allelic variants of three genes associated with adverse reactions to common chemotherapeutic agents: DPYD (Dihydropyrimidine dehydrogenase), MTHFR (5,10-Methylenetetrahydrofolate reductase) and TPMT (Thiopurine S-methyltransferase). Results: Inter- and intra assay variability were performed to assess the specificity and sensibility of the chip. Linear regression was used to assess the optimal hybridization temperature set at 52 °C (R2 ≈ 0.97). Limit of detection was 50 nM. Conclusions: The high performance in terms of sensibility and specificity of this lab-on-chip supports its further translation to clinical diagnostics, where it may effectively promote precision medicine.

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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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Therapeutic Drug Monitoring: Performance of a FRET-Based Point-Of-Care Immunoassay for the Quantitation of Infliximab and Adalimumab in Blood

10.26434/chemrxiv.13277321.v1 ◽

2020 ◽

Author(s):

Edgar Ong ◽

Ruo Huang ◽

Richard Kirkland ◽

Stefan Westin ◽

Jared Salbato ◽

...

Keyword(s):

Therapeutic Drug Monitoring ◽

Drug Monitoring ◽

Whole Blood ◽

Point Of Care ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Drug Level ◽

Analytical Performance ◽

Quantitative Detection ◽

Therapeutic Drug

Two fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassays (Procise IFX™ and Procise ADL™) were developed for the quantitative detection of infliximab (IFX), adalimumab (ADL), and their respective biosimilars for use in therapeutic drug monitoring (TDM) using 20 µL of finger prick whole blood at the point-of-care or whole blood/serum in a central lab. Studies were performed to characterize analytical performance of the Procise IFX and the Procise ADL assays on the ProciseDx™ analyzer. The Procise IFX and Procise ADL assays both showed good analytical performance with respect to sensitivity, specificity, linearity, and precision suitable for routine clinical use as well as excellent correlation to current commercial ELISA IFX and ADL measurement methods. Results indicated that the Procise IFX and Procise ADL assays are sensitive, specific, and precise yielding results in less than 5 minutes from either whole blood or serum. This indicates the Procise IFX and Procise ADL assays are useful for obtaining fast and accurate IFX or ADL quantitation, thus avoiding delays inherent to current methods and enabling immediate drug level dosing decisions to be made during a single patient visit.

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