Miniaturized Multiplex Label-Free Electronic Chip for Rapid Nucleic Acid Analysis Based on Carbon Nanotube Nanoelectrode Arrays

Abstract Background: Reducing cost and time is the major concern in clinical diagnostics, particularly in molecular diagnostics. Miniaturization technologies have been recognized as promising solutions to provide low-cost microchips for diagnostics. With the recent advancement in nanotechnologies, it is possible to further improve detection sensitivity and simplify sample preparation by incorporating nanoscale elements in diagnostics devices. A fusion of micro- and nanotechnologies with biology has great potential for the development of low-cost disposable chips for rapid molecular analysis that can be carried out with simple handheld devices. Approach: Vertically aligned multiwalled carbon nanotubes (MWNTs) are fabricated on predeposited microelectrode pads and encapsulated in SiO2 dielectrics with only the very end exposed at the surface to form an inlaid nanoelectrode array (NEA). The NEA is used to collect the electrochemical signal associated with the target molecules binding to the probe molecules, which are covalently attached to the end of the MWNTs. Content: A 3 × 3 microelectrode array is presented to demonstrate the miniaturization and multiplexing capability. A randomly distributed MWNT NEA is fabricated on each microelectrode pad. Selective functionalization of the MWNT end with a specific oligonucleotide probe and passivation of the SiO2 surface with ethylene glycol moieties are discussed. Ru(bpy)2+-mediator-amplified guanine oxidation is used to directly measure the electrochemical signal associated with target molecules. Summary: The discussed MWNT NEAs have ultrahigh sensitivity in direct electrochemical detection of guanine bases in the nucleic acid target. Fewer than ∼1000 target nucleic acid molecules can be measured with a single microelectrode pad of ∼20 × 20 μm2, which approaches the detection limit of laser scanners in fluorescence-based DNA microarray techniques. MWNT NEAs can be easily integrated with microelectronic circuitry and microfluidics for development of a fully automated system for rapid molecular analysis with minimum cost.

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Recent Advances in Macromolecularly Imprinted Polymers by Controlled Radical Polymerization Techniques

Molecular Imprinting ◽

10.1515/molim-2015-0005 ◽

2015 ◽

Vol 3 (1) ◽

Cited By ~ 2

Author(s):

Huiqi Zhang

Keyword(s):

Radical Polymerization ◽

Low Cost ◽

Proteome Analysis ◽

Controlled Radical Polymerization ◽

Clinical Diagnostics ◽

Imprinted Polymers ◽

Recognition Ability ◽

Recent Developments ◽

Target Molecules ◽

Controlled Preparation

AbstractMolecularly imprinted polymers (MIPs) are synthetic receptors with tailor-made recognition sites for the target molecules. Their high molecular recognition ability, good stability, easy preparation, and low cost make them highly promising substitutes for biological receptors. Recent years have witnessed rapidly increasing interest in the imprinting of biomacromolecules and especially proteins because of the great potential of these MIPs in such applications as proteome analysis, clinical diagnostics, and biomedicine. So far, some useful strategies have been developed for the imprinting of proteins and controlled radical polymerization techniques have proven highly versatile for such purpose. This mini-review describes recent developments in the controlled preparation of proteins-imprinted polymers via such advanced polymerization techniques.

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CRISPR/Cas13a powered electrochemical microfluidic biosensor for nucleic acid amplification-free miRNA diagnostics

10.1101/738617 ◽

2019 ◽

Author(s):

Richard Bruch ◽

Julia Baaske ◽

Claire Chatelle ◽

Mailin Meirich ◽

Sibylle Madlener ◽

...

Keyword(s):

Nucleic Acid ◽

Limit Of Detection ◽

Early Stage ◽

Low Cost ◽

Clinical Diagnostics ◽

Nucleic Acid Amplification ◽

Process Time ◽

Serum Samples ◽

Microfluidic Biosensor ◽

Pcr Method

Non-coding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated to many different diseases, such as cancer, dementia or cardiovascular conditions. The key for an effective treatment is an accurate initial diagnosis at an early stage, improving the patient’s survival chances. Here, we introduce a CRISPR/Cas13a powered microfluidic, integrated electrochemical biosensor for the on-site detection of microRNAs. Through this unique combination, the quantification of the potential tumor markers microRNA miR-19b and miR-20a has been realized without any nucleic acid amplification. With a readout time of 9 minutes and an overall process time of less than 4 hours, a limit of detection of 10 pM was achieved, using a measuring volume of less than 0.6 µl. Furthermore, we demonstrate the feasibility of our versatile sensor platform to detect miR-19b in serum samples of children, suffering from brain cancer. The validation of our results with a standard qRT-PCR method shows the ability of our system to be a low-cost and target amplification-free tool for nucleic acid based diagnostics.

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Label-Free Detection of Zeptomol miRNA via Peptide Nucleic Acid Hybridization Using Novel Cyclic Voltammetry Method

Sensors ◽

10.3390/s20030836 ◽

2020 ◽

Vol 20 (3) ◽

pp. 836

Author(s):

Shintaro Takase ◽

Kouta Miyagawa ◽

Hisafumi Ikeda

Keyword(s):

Nucleic Acid ◽

Peptide Nucleic Acid ◽

Microelectrode Array ◽

Pcr Amplification ◽

Nucleic Acid Hybridization ◽

Detection Sensitivity ◽

Cyclic Voltammograms ◽

Dna Duplexes ◽

Label Free ◽

Label Free Detection

To harness the applicability of microribonucleic acid (miRNA) as a cancer biomarker, the detection sensitivity of serum miRNA needs to be improved. This study evaluated the detection sensitivity of miRNA hybridization using cyclic voltammograms (CVs) and microelectrode array chips modified with peptide nucleic acid (PNA) probes and 6-hydroxy-1-hexanethiol. We investigated the PNA probe modification pattern on array chips using fluorescently labeled cDNA. The pattern was not uniformly spread over the working electrode (WE) and had a one-dimensional swirl-like pattern. Accordingly, we established a new ion-channel sensor model wherein the WE is negatively biased through the conductive π–π stacks of the PNA/DNA duplexes. This paper discusses the mechanism underlying the voltage shift in the CV curves based on the electric double-layer capacitance. Additionally, the novel hybridization evaluation parameter ΔE is introduced. Compared to conventional evaluation using oxidation current changes, ΔE was more sensitive. Using ΔE and a new hybridization system for ultrasmall amounts of aqueous solutions (as low as 35 pL), 140 zeptomol label-free miRNA were detected without polymerase chain reaction (PCR) amplification at an adequate sensitivity. Herein, the differences in the target molar amount and molar concentration are elucidated from the viewpoint of hybridization sensitivity.

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Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

Frontiers in Microbiology ◽

10.3389/fmicb.2021.751408 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jian Zhang ◽

Hailong Lv ◽

Linxian Li ◽

Minjie Chen ◽

Dayong Gu ◽

...

Keyword(s):

Nucleic Acid ◽

Pathogen Detection ◽

Direct Detection ◽

Detection Sensitivity ◽

Molecular Diagnostic ◽

Target Nucleic Acid ◽

Precise Diagnosis ◽

Important Approach ◽

Pathogen Diagnosis ◽

Diagnostic Technologies

Molecular diagnostic (MDx) methods directly detect target nucleic acid sequences and are therefore an important approach for precise diagnosis of pathogen infection. In comparison with traditional MDx techniques such as PCR, the recently developed CRISPR-based diagnostic technologies, which employ the single-stranded nucleic acid trans-cleavage activities of either Cas12 or Cas13, show merits in both sensitivity and specificity and therefore have great potential in both pathogen detection and beyond. With more and more efforts in improving both the CRISPR trans-cleavage efficiencies and the signal detection sensitivities, CRISPR-based direct detection of target nucleic acids without preamplification can be a possibility. Here in this mini-review, we summarize recent research progresses of amplification-free CRISPR-Dx systems and explore the potential changes they will lead to pathogen diagnosis. In addition, discussion of the challenges for both detection sensitivity and cost of the amplification-free systems will also be covered.

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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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A glucose meter interface for point-of-care gene circuit-based diagnostics

Nature Communications ◽

10.1038/s41467-020-20639-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Evan Amalfitano ◽

Margot Karlikow ◽

Masoud Norouzi ◽

Katariina Jaenes ◽

Seray Cicek ◽

...

Keyword(s):

Nucleic Acid ◽

Synthetic Biology ◽

Molecular Diagnostics ◽

Point Of Care ◽

Low Cost ◽

Gene Circuit ◽

Reporter Systems ◽

Glucose Meter ◽

Pandemic Response ◽

Glucose Output

AbstractRecent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

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Nucleic-Acid-Binding Chromophores as Efficient Indicators of Aptamer-Target Interactions

Journal of Nucleic Acids ◽

10.1155/2012/247280 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 8

Author(s):

Kwabena Sarpong ◽

Bhaskar Datta

Keyword(s):

Nucleic Acid ◽

High Sensitivity ◽

Covalent Modification ◽

Fluorescence Signal ◽

Nucleic Acid Binding ◽

Sensitivity And Selectivity ◽

Target Molecules ◽

Diagnostic Applications ◽

The Stability ◽

Simple Format

The binding affinity and specificity of nucleic acid aptamers have made them valuable candidates for use as sensors in diagnostic applications. In particular, chromophore-functionalized aptamers offer a relatively simple format for detection and quantification of target molecules. We describe the use of nucleic-acid-staining reagents as an effective tool for detecting and signaling aptamer-target interactions. Aptamers varying in size and structure and targeting a range of molecules have been used in conjunction with commercially available chromophores to indicate and quantify the presence of cognate targets with high sensitivity and selectivity. Our assay precludes the covalent modification of nucleic acids and relies on the differential fluorescence signal of chromophores when complexed with aptamers with or without their cognate target. We also evaluate factors that are critical for the stability of the complex between the aptamer and chromophore in presence or absence of target molecules. Our results indicate the possibility of controlling those factors to enhance the sensitivity of target detection by the aptamers used in such assays.

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Automated text messaging follow-up for patients who receive peripheral nerve blocks

Regional Anesthesia and Pain Medicine ◽

10.1136/rapm-2021-102472 ◽

2021 ◽

pp. rapm-2021-102472

Author(s):

Daniel Gessner ◽

Oluwatobi O Hunter ◽

Alex Kou ◽

Edward R Mariano

Keyword(s):

Regional Anesthesia ◽

Nerve Block ◽

Text Messaging ◽

Low Cost ◽

Automated System ◽

Phone Call ◽

Nerve Blocks ◽

Patient Response ◽

Patient Reported

BackgroundRoutine follow-up of patients who receive a nerve block for ambulatory surgery typically consists of a phone call from a regional anesthesia clinician. This process can be burdensome for both patients and clinicians but is necessary to assess the efficacy and complication rate of nerve blocks.MethodsWe present our experience developing an automated system for completing follow-up via short message service text messaging and our preliminary results using it at three clinical sites. The system is built on REDCap, a secure online research data capture platform developed by Vanderbilt University and currently available worldwide.ResultsOur automated system queried patients who received a variety of nerve block techniques, assessed patient-reported nerve block duration, and surveyed patients for potential complications. Patient response rate to text messaging averaged 91% (higher than our rates of daily phone contact reported previously) for patients aged 18 to 90 years.ConclusionsGiven the wide availability of REDCap, we believe this automated text messaging system can be implemented in a variety of health systems at low cost with minimal technical expertise and will improve both the consistency of patient follow-up and the service efficiency of regional anesthesia practices.

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