Integrated, Automated, Fast PCR System for Point-Of-Care Molecular Diagnosis of Bacterial Infection

We developed an integrated PCR system that performs automated sample preparation and fast polymerase chain reaction (PCR) for application in point-of care (POC) testing. This system is assembled from inexpensive 3D-printing parts, off-the-shelf electronics and motors. Molecular detection requires a series of procedures including sample preparation, amplification, and fluorescence intensity analysis. The system can perform automated DNA sample preparation (extraction, separation and purification) in ≤5 min. The variance of the automated sample preparation was clearly lower than that achieved using manual DNA extraction. Fast thermal ramp cycles were generated by a customized thermocycler designed to automatically transport samples between heating and cooling blocks. Despite the large sample volume (50 μL), rapid two-step PCR amplification completed 40 cycles in ≤13.8 min. Variations in fluorescence intensity were measured by analyzing fluorescence images. As proof of concept of this system, we demonstrated the rapid DNA detection of pathogenic bacteria. We also compared the sensitivity of this system with that of a commercial device during the automated extraction and fast PCR of Salmonella bacteria.

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A plasmonic gold nanofilm-based microfluidic chip for rapid and inexpensive droplet-based photonic PCR

Scientific Reports ◽

10.1038/s41598-021-02535-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Abbas Jalili ◽

Maryam Bagheri ◽

Amir Shamloo ◽

Amir Hossein Kazemipour Ashkezari

Keyword(s):

Microfluidic Chip ◽

Point Of Care ◽

Pcr Amplification ◽

Alcohol Oxidase ◽

Nucleic Acid Amplification ◽

Adhesive Tape ◽

Microfluidic Chips ◽

Fast Heating ◽

Light Emitting ◽

Heating And Cooling

AbstractPolymerase chain reaction (PCR) is a powerful tool for nucleic acid amplification and quantification. However, long thermocycling time is a major limitation of the commercial PCR devices in the point-of-care (POC). Herein, we have developed a rapid droplet-based photonic PCR (dpPCR) system, including a gold (Au) nanofilm-based microfluidic chip and a plasmonic photothermal cycler. The chip is fabricated by adding mineral oil to uncured polydimethylsiloxane (PDMS) to suppress droplet evaporation in PDMS microfluidic chips during PCR thermocycling. A PDMS to gold bonding technique using a double-sided adhesive tape is applied to enhance the bonding strength between the oil-added PDMS and the gold nanofilm. Moreover, the gold nanofilm excited by two light-emitting diodes (LEDs) from the top and bottom sides of the chip provides fast heating of the PCR sample to 230 °C within 100 s. Such a design enables 30 thermal cycles from 60 to 95 °C within 13 min with the average heating and cooling rates of 7.37 ± 0.27 °C/s and 1.91 ± 0.03 °C/s, respectively. The experimental results demonstrate successful PCR amplification of the alcohol oxidase (AOX) gene using the rapid plasmonic photothermal cycler and exhibit the great performance of the microfluidic chip for droplet-based PCR.

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A robust, hand-powered, instrument-free sample preparation system for point-of-care pathogen detection

Scientific Reports ◽

10.1038/s41598-019-52922-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Fei Zhao ◽

Eun Yeong Lee ◽

Geun Su Noh ◽

Jaehyup Shin ◽

Huifang Liu ◽

...

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Pathogen Detection ◽

Point Of Care ◽

Low Cost ◽

Sample Pretreatment ◽

Sample Volume ◽

Nucleic Acid Isolation ◽

Point Of Care Diagnostics ◽

Reaction Matrix

Abstract Here, we describe a simple, universal protocol for use in nucleic acid testing-based pathogen diagnostics, which requires only hand-powered sample preparation, including the processes of pathogen enrichment and nucleic acid isolation. The protocol uses low-cost amine-functionalized diatomaceous earth with a 1-μm Teflon filter as a reaction matrix in both stages of the process, using homobifunctional imidoesters. Using a simple syringe as a pump, the capture efficiency for a large sample volume (<50 mL) was enhanced by up to 98.3%, and the detection limit was 1 CFU/mL, 100-fold better than that of common commercial nucleic acid isolation kit. This protocol can also be combined with commercialized 96-well filter plates for robust sample preparation. Our proposed system is robust, simple, low-cost, universal, and rapid (taking <20 min), and it works regardless of the ambient environment and sample pretreatment, requiring no electricity or instruments. Its benefits include the simplicity of producing its components and its ease of operation, and it can be readily integrated with other assays for point-of-care diagnostics.

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Automated Blood Sample Preparation Unit (ABSPU) for Portable Microfluidic Flow Cytometry

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2211068216663604 ◽

2016 ◽

Vol 22 (1) ◽

pp. 73-80 ◽

Cited By ~ 2

Author(s):

Akhil Chaturvedi ◽

Sai Siva Gorthi

Keyword(s):

Sample Preparation ◽

Blood Sample ◽

Point Of Care ◽

Low Cost ◽

Flow Cytometric Analysis ◽

Cell Integrity ◽

Microfluidic Flow ◽

Out Of Sample ◽

Microfluidic Analysis ◽

Automated Sample Preparation

Portable microfluidic diagnostic devices, including flow cytometers, are being developed for point-of-care settings, especially in conjunction with inexpensive imaging devices such as mobile phone cameras. However, two pervasive drawbacks of these have been the lack of automated sample preparation processes and cells settling out of sample suspensions, leading to inaccurate results. We report an automated blood sample preparation unit (ABSPU) to prevent blood samples from settling in a reservoir during loading of samples in flow cytometers. This apparatus automates the preanalytical steps of dilution and staining of blood cells prior to microfluidic loading. It employs an assembly with a miniature vibration motor to drive turbulence in a sample reservoir. To validate performance of this system, we present experimental evidence demonstrating prevention of blood cell settling, cell integrity, and staining of cells prior to flow cytometric analysis. This setup is further integrated with a microfluidic imaging flow cytometer to investigate cell count variability. With no need for prior sample preparation, a drop of whole blood can be directly introduced to the setup without premixing with buffers manually. Our results show that integration of this assembly with microfluidic analysis provides a competent automation tool for low-cost point-of-care blood-based diagnostics.

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Automated Sample Preparation (ASP): Development of a Rapid Method to Sequentially Isolate Nucleic Acids and Protein from Any Sample Type by a Cartridge-Based System

10.21236/ada608052 ◽

2013 ◽

Author(s):

David R. Pawlowski

Keyword(s):

Sample Preparation ◽

Nucleic Acids ◽

Rapid Method ◽

Sample Type ◽

Automated Sample Preparation

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Comparison of 2 glucose analytical methodologies in immature Kemp’s ridley sea turtles: dry chemistry of plasma versus point-of-care glucometer analysis of whole blood

Journal of Veterinary Diagnostic Investigation ◽

10.1177/10406387211001830 ◽

2021 ◽

pp. 104063872110018

Author(s):

Justin R. Perrault ◽

Michael D. Arendt ◽

Jeffrey A. Schwenter ◽

Julia L. Byrd ◽

Kathryn A. Tuxbury ◽

...

Keyword(s):

Whole Blood ◽

Point Of Care ◽

Sea Turtles ◽

Sample Volume ◽

Specific Reference ◽

Volume Data ◽

Analytical Methodology ◽

Glucose Determination ◽

Kemp's Ridley Sea Turtles ◽

Kemp's Ridley

Blood glucose measurements provide important diagnostic information regarding stress, disease, and nutritional status. Glucose analytical methodologies include dry chemistry analysis (DCA) of plasma and point-of-care (POC) glucometer analysis of whole blood; however, these 2 methods differ in cost, required sample volume, and processing time. Because POC glucometers use built-in equations based on features of mammalian blood to convert whole blood measurements to plasma equivalent units, obtained glucose data must be compared and validated using gold-standard chemistry analytical methodology in reptiles. For in-water, trawl-captured, immature Kemp’s ridley sea turtles ( Lepidochelys kempii) from Georgia, USA, we observed significant, positive agreement between the 2 glucose determination methods; however, the glucometer overestimated glucose concentrations by 1.4 mmol/L on average in comparison to DCA and produced a wider range of results. The discordance of these results suggests that POC glucometer glucose data should be interpreted in the context of methodology- and brand-specific reference intervals along with concurrent packed cell volume data.

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Automated sample preparation and analysis by gas chromatography tandem mass spectrometry (GC-MS/MS) for the determination of 3- and 2-monochloropropanediol (MCPD) esters and glycidol esters in edible oils

Journal of Chromatography A ◽

10.1016/j.chroma.2021.462253 ◽

2021 ◽

pp. 462253

Author(s):

Pasquale Ioime ◽

Elena Piva ◽

Michele Pozzebon ◽

Jennifer P. Pascali

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Sample Preparation ◽

Tandem Mass Spectrometry ◽

Edible Oils ◽

Tandem Mass ◽

Chromatography Tandem Mass Spectrometry ◽

Mcpd Esters ◽

Automated Sample Preparation

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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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A tandem giant magnetoresistance assay for one-shot quantification of clinically relevant concentrations of N-terminal pro-B-type natriuretic peptide in human blood

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03227-5 ◽

2021 ◽

Author(s):

Fanda Meng ◽

Weisong Huo ◽

Jie Lian ◽

Lei Zhang ◽

Xizeng Shi ◽

...

Keyword(s):

Detection Limit ◽

Giant Magnetoresistance ◽

Dynamic Range ◽

Point Of Care ◽

Sample Volume ◽

Small Sample ◽

Broad Dynamic Range ◽

Gmr Sensors ◽

Gmr Sensor ◽

Sample Volume Requirement

AbstractWe report a microfluidic sandwich immunoassay constructed around a dual-giant magnetoresistance (GMR) sensor array to quantify the heart failure biomarker NT-proBNP in human plasma at the clinically relevant concentration levels between 15 pg/mL and 40 ng/mL. The broad dynamic range was achieved by differential coating of two identical GMR sensors operated in tandem, and combining two standard curves. The detection limit was determined as 5 pg/mL. The assay, involving 53 plasma samples from patients with different cardiovascular diseases, was validated against the Roche Cobas e411 analyzer. The salient features of this system are its wide concentration range, low detection limit, small sample volume requirement (50 μL), and the need for a short measurement time of 15 min, making it a prospective candidate for practical use in point of care analysis.

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Nitrocellulose-bound achromopeptidase for point-of-care nucleic acid tests

Scientific Reports ◽

10.1038/s41598-021-85481-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Georgios Chondrogiannis ◽

Shirin Khaliliazar ◽

Anna Toldrà ◽

Pedro Réu ◽

Mahiar M. Hamedi

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Point Of Care ◽

Dna Amplification ◽

Recombinase Polymerase Amplification ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Enzymatic Function ◽

Modern Biotechnology ◽

Nucleic Acid Tests

AbstractEnzymes are the cornerstone of modern biotechnology. Achromopeptidase (ACP) is a well-known enzyme that hydrolyzes a number of proteins, notably proteins on the surface of Gram-positive bacteria. It is therefore used for sample preparation in nucleic acid tests. However, ACP inhibits DNA amplification which makes its integration difficult. Heat is commonly used to inactivate ACP, but it can be challenging to integrate heating into point-of-care devices. Here, we use recombinase polymerase amplification (RPA) together with ACP, and show that when ACP is immobilized on nitrocellulose paper, it retains its enzymatic function and can easily and rapidly be activated using agitation. The nitrocellulose-bound ACP does, however, not leak into the solution, preventing the need for deactivation through heat or by other means. Nitrocellulose-bound ACP thus opens new possibilities for paper-based Point-of-Care (POC) devices.

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