MICROFLUIDIC FLOW-THROUGH REACTOR WITH ELECTROCHEMICAL SENSOR ARRAY FOR REAL-TIME PCR

2009 ◽  
Vol 23 (03) ◽  
pp. 369-372
Author(s):  
HUEY-FANG TEH ◽  
NAVEEN RAMALINGAM ◽  
HAI-QING GONG ◽  
SWEE-NGIN TAN
Keyword(s):  
Real Time ◽  
Fluorescence Detection ◽  
Microelectrode Array ◽  
Dna Amplification ◽  
Active Species ◽  
Dna Intercalator ◽  
Detection Scheme ◽  
Microfluidic Flow ◽  
Pcr Products ◽  
Flow Through

We developed an integrated microfluidic flow-through EC-PCR (EC-PCR) microdevice for the concurrent DNA amplification, PCR products EC detection and PCR products quantification instead of the current available fluorescence detection scheme. The microfluidic flow-through EC-PCR microdevice was fabricated with the state-of-the-art microfabrication technology, by bonding a bottom glass substrate having a microelectrode array to a top glass cover having the microchannels made of PDMS material. Both the amplification of the target DNA sequence and the subsequent EC detection of the PCR products were carried out concurrently on the integrated device by real-time monitoring. The underlying principle of the microfluidic flow-through EC-PCR method was based on the changes of current signal of methylene blue (MB), which worked as an electrochemically active species DNA intercalator in the PCR mixture, during the amplification process at the extension phase. The results shown in this work indicated that the nucleic acid analysis could be performed in a fast thermal cycling and true real-time quantitative electrochemical detection. The signal variation trends of the EC detection and the fluorescence detection were the same in our verification measurements for both methods, which suggested that the EC detection method was feasible for this application.

scholarly journals Multiple Camera Fluorescence Detection for Real-Time PCR

2021 ◽  
Vol 6 (1) ◽  
pp. 71
Author(s):  
Seul-Bit-Na Koo ◽  
Hyeon-Gyu Chi ◽  
Ji-Sung Park ◽  
Jong-Dae Kim ◽  
Chan-Young Park ◽  
...  
Keyword(s):  
Real Time ◽  
Fluorescence Detection ◽  
Real Time Pcr ◽  
Detection System ◽  
Low Cost ◽  
Dna Amplification ◽  
Multiple Camera ◽  
Open Platform ◽  
Polymerase Chain ◽  
Fluorescence Detection System

The general polymerase chain reaction (PCR) amplifies DNA and analyzes the amplification results of the quantified DNA. Recently, real-time PCR has been developed to detect DNA amplification in various ways. The conventional camera-based system is too expensive and difficult to reduce device size. In this paper, we propose a low-cost, compact fluorescence detection system for real-time PCR systems using an open platform camera. To simplify the optics, four low-cost small cameras were fixedly placed, and the entire tube was divided into four quadrants to minimize the field of view. In addition, an effective image processing method was used to compensate. The proposed system measured the fluorescence detection performance on the basis of the amount of DNA using various fluorescent substances.

scholarly journals Electrokinetic Formation of “Microbridges” for Protein Biomarkers as Sensors

2007 ◽  
Vol 12 (5) ◽  
pp. 311-317 ◽  
Author(s):  
Vindhya Kunduru ◽  
Shalini Prasad
Keyword(s):  
Electric Fields ◽  
Microelectrode Array ◽  
Protein Detection ◽  
High Specificity ◽  
Detection Methods ◽  
Label Free ◽  
Protein Biomarkers ◽  
Detection Scheme ◽  
Polystyrene Beads ◽  
Conducting Path

We demonstrate a technique to detect protein biomarkers contained in vulnerable coronary plaque using a platform-based microelectrode array (MEA). The detection scheme is based on the property of high specificity binding between antibody and antigen similar to most immunoassay techniques. Rapid clinical diagnosis can be achieved by detecting the amount of protein in blood by analyzing the protein's electrical signature. Polystyrene beads which act as transportation agents for the immobile proteins (antigen) are electrically aligned by application of homogenous electric fields. The principle of electrophoresis is used to produce calculated electrokinetic movement among the anti-C-reactive protein (CRP), or in other words antibody funtionalized polystyrene beads. The electrophoretic movement of antibody-functionalized polystyrene beads results in the formation of “Microbridges” between the two electrodes of interest which aid in the amplification of the antigen—antibody binding event. Sensitive electrical equipment is used for capturing the amplified signal from the “Microbridge” which essentially behaves as a conducting path between the two electrodes. The technique circumvents the disadvantages of conventional protein detection methods by being rapid, noninvasive, label-free, repeatable, and inexpensive. The same principle of detection can be applied for any receptor—ligand-based system because the technique is based only on the volume of the analyte of interest. Detection of the inflammatory coronary disease biomarker CRP is achieved at concentration levels spanning over the lower microgram/milliliter to higher order nanogram/milliliter ranges.

A novel microfluidic microelectrode chip for a significantly enhanced monitoring of NPY-receptor activation in live mode

Lab on a Chip ◽  
2017 ◽  
Vol 17 (24) ◽  
pp. 4294-4302 ◽  
Author(s):  
Franziska D. Zitzmann ◽  
Heinz-Georg Jahnke ◽  
Felix Nitschke ◽  
Annette G. Beck-Sickinger ◽  
Bernd Abel ◽  
...  
Keyword(s):  
Real Time ◽  
Microfluidic Chip ◽  
Microelectrode Array ◽  
Fem Simulation ◽  
Receptor Activation ◽  
Living Cells ◽  
Real Time Monitoring ◽  
Non Invasive ◽  
Npy Receptor ◽  
Simulation Based

We present a FEM simulation based step-by-step development of a microelectrode array integrated into a microfluidic chip for the non-invasive real-time monitoring of living cells.

scholarly journals Quantitative Analysis of Fluorescence Detection Using a Smartphone Camera for a PCR Chip

Sensors ◽  
2021 ◽  
Vol 21 (11) ◽  
pp. 3917
Author(s):  
Jong-Dae Kim ◽  
Chan-Young Park ◽  
Yu-Seop Kim ◽  
Ji-Soo Hwang
Keyword(s):  
Quantitative Analysis ◽  
Fluorescence Detection ◽  
High Performance ◽  
Low Cost ◽  
Dna Amplification ◽  
Fluorescent Detection ◽  
Rt Pcr ◽  
Commercial System ◽  
Intensity Changes ◽  
The Difference

Most existing commercial real-time polymerase chain reaction (RT-PCR) instruments are bulky because they contain expensive fluorescent detection sensors or complex optical structures. In this paper, we propose an RT-PCR system using a camera module for smartphones that is an ultra small, high-performance and low-cost sensor for fluorescence detection. The proposed system provides stable DNA amplification. A quantitative analysis of fluorescence intensity changes shows the camera’s performance compared with that of commercial instruments. Changes in the performance between the experiments and the sets were also observed based on the threshold cycle values in a commercial RT-PCR system. The overall difference in the measured threshold cycles between the commercial system and the proposed camera was only 0.76 cycles, verifying the performance of the proposed system. The set calibration even reduced the difference to 0.41 cycles, which was less than the experimental variation in the commercial system, and there was no difference in performance.

Diagnosis and Causative Species of Visceral Leishmaniasis in Southwest Saudi Arabia

2021 ◽  
Author(s):  
Aymen Abdelhaleem ◽  
Nabil Dhayhi ◽  
Mohamed Salih Mahfouz ◽  
Ommer Daffalla ◽  
Mansour Mubarki ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Visceral Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Donovani ◽  
Target Genes ◽  
Sample Volume ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Robust Evidence

Visceral leishmaniasis (VL) is the most severe clinical form of the disease and has been reported in the Jazan region of southwest Saudi Arabia. This study aimed to diagnose VL by real-time polymerase chain reaction (PCR) and the direct agglutination test (DAT) and to identify the causative Leishmania species. A total of 80 participants, including 30 suspected VL patients, 30 healthy endemic control individuals, and 20 malaria disease controls, were enrolled in this study. Blood samples were collected and tested for Leishmania DNA by real-time PCR and for antibody by the DAT. Sequencing of some amplified PCR products was used to identify the causative Leishmania species. The diagnosis of VL was successfully achieved by both real-time PCR and by DAT with 100% sensitivity. Leishmania donovani and Leishmania infantum species were detected by sequencing both by the kDNA and ITS1 target genes, followed a BLASTn search. The detection of VL antibody by the DAT followed by the confirmatory detection of Leishmania DNA in patient blood by PCR could promote the adoption of the much less invasive and more sensitive methods for the routine diagnosis of VL. Further study with high sample volume to evaluate the PCR and the DAT are needed, to generate more robust evidence. Based on the sequencing results, emerging studies on VL should focus on the causative Leishmania species, reservoirs, and vectors that are important in the study area.

scholarly journals Integrated Extreme Real-Time PCR and High-Speed Melting Analysis in 52 to 87 Seconds

2019 ◽  
Vol 65 (2) ◽  
pp. 263-271 ◽  
Author(s):  
Joseph T Myrick ◽  
Robert J Pryor ◽  
Robert A Palais ◽  
Sean J Ison ◽  
Lindsay Sanford ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Speed ◽  
Dna Analysis ◽  
Point Of Care ◽  
Turnaround Time ◽  
Single Nucleotide Variants ◽  
Cycle Times ◽  
Pcr Products ◽  
Melting Analysis

Abstract BACKGROUND Extreme PCR in <30 s and high-speed melting of PCR products in <5 s are recent advances in the turnaround time of DNA analysis. Previously, these steps had been performed on different specialized instruments. Integration of both extreme PCR and high-speed melting with real-time fluorescence monitoring for detection and genotyping is presented here. METHODS A microfluidic platform was enhanced for speed using cycle times as fast as 1.05 s between 66.4 °C and 93.7 °C, with end point melting rates of 8 °C/s. Primer and polymerase concentrations were increased to allow short cycle times. Synthetic sequences were used to amplify fragments of hepatitis B virus (70 bp) and Clostridium difficile (83 bp) by real-time PCR and high-speed melting on the same instrument. A blinded genotyping study of 30 human genomic samples at F2 c.*97, F5 c.1601, MTHFR c.665, and MTHFR c.1286 was also performed. RESULTS Standard rapid-cycle PCR chemistry did not produce any product when total cycling times were reduced to <1 min. However, efficient amplification was possible with increased primer (5 μmol/L) and polymerase (0.45 U/μL) concentrations. Infectious targets were amplified and identified in 52 to 71 s. Real-time PCR and genotyping of single-nucleotide variants from human DNA was achieved in 75 to 87 s and was 100% concordant to known genotypes. CONCLUSIONS Extreme PCR with high-speed melting can be performed in about 1 min. The integration of extreme PCR and high-speed melting shows that future molecular assays at the point of care for identification, quantification, and variant typing are feasible.

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