ArticlePublisher preview available

An automated microfluidic chemiluminescence immunoassay platform for quantitative detection of biomarkers

October 2018
Biomedical Microdevices 20(4)

October 2018
20(4)

DOI:10.1007/s10544-018-0331-3

Authors:

Jianzhong Zhang

Northwestern University

Show all 11 authorsHide

A rapid, sensitive and quantitative biomarker detection platform is of great importance to the small clinic or point-of-care (POC) diagnosis. In this work, we realize that an automated diagnostic platform mainly includes two components: (1) an instrument that can complete all steps of the chemiluminescence immunoassay automatically and (2) an integrated microfluidic chip which is disposable and harmless. In the instrument, we adopt vacuum suction cups which are driven by linear motor to realize a simple, effective and convenient control. The method of acridine esterification chemiluminescence is adopted to achieve a quantitative detection, and a photomultiplier tube is used to detect photons from acridine ester producing in alkaline conditions. We use the laser cutting machine and hot press machine to accomplish the product of microfluidic chips. The automated microfluidics-based system is demonstrated by implementation of a chemiluminescence immunoassay for quantitative detection of ferritin. We observe alinear relationship between CL intensity and the concentration of ferritin from 5.1 to 1300 ng mL ⁻¹and the limit of detection (LoD) is 2.55 ng mL ⁻¹. At the same time, we also used the automated microfluidics-based system to test clinical serum samples. The whole process of chemiluminescence experiment can complete within 45 min. We realize that this lab-on-a-chip chemiluminescence immunoassay platform with features of automation and quantitation provides a promising strategy for POC diagnosis.

Design and assembly of an integrated microfluidic chip for chemiluminescence immunoassay. (a) Schematic of the top layer with different holes, the middle layer with embedded microchannels and reservoirs, and the bottom silicone substrate layer before assembly (b) Photograph of the assembled microfluidic chip

…

(a) Design and photograph of the portable and automated instrument for chip-based chemiluminescence immunoassay. (b) The main components of the instrument include a photomultiplier tube, vacuum suction cup, circuit board, peristaltic pump, and negative pressure connector

…

Theautomatical experiment of reagents reaction in the chip (a) Positive pressure opening of the second reservoir drive ferritin sample and magnetic beads pre-coated with Ferritin-Ab1 crossing the microchannel together from the reservoir into the reaction chamber. (b) Peristaltic pump provides negative pressure to blow the hole and realize the reagent from reaction reservoir to waste liquid reservoir. (c) Peristaltic pump provides positive pressure to blow the hole and the mixture in the reaction chamber gets bubbles

…

The state of the magnetic beads in different reaction stages (a)The magnetic beads are mixed by the bubbles (b) The magnetic beads are adsorbed and fixed in the chamber by the magnets

…

Schematic of microfluidic chemiluminescence immunoassay for ferritin detection. Workflow of CRP detection (a) The magnetic particles pre-coated with Ferritin-Ab1and ferritin are in incubation for 15 min (b) The combination is washed by wash buffer (each 120ul) for two times. (c) Acridinium ester marked Ferritin-Ab2 from the reservoir into the reaction chamber, blending with the combination, and then incubation for 10 min.(d) The combination is washed by wash buffer(each 120ul) for two times again.(e) PTS and TS are introduced into reaction reservoir and generate the CL signal

…

Figures - available from: Biomedical Microdevices

This content is subject to copyright. Terms and conditions apply.

A preview of this full-text is provided by Springer Nature.

Learn more

Content available from Biomedical Microdevices

This content is subject to copyright. Terms and conditions apply.

An automated microfluidic chemiluminescence immunoassay platform

for quantitative detection of biomarkers

Xiaoping Min

1,2,3

&Da Fu

2,4

&Jianzhong Zhang

2,4

&Juntian Zeng

&Zhenyu Weng

2,4

&Wendi Chen

&Shiyin Zhang

2,3,4

Dongxu Zhang

2,3,4

&Shengxiang Ge

2,3,4

&Jun Zhang

2,3,4

&Ningshao Xia

2,3,4

Published online: 25 October 2018

#Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract

A rapid, sensitive and quantitative biomarker detection platform is of great importance to the small clinic or point-of-care (POC)

diagnosis. In this work, we realize that an automated diagnostic platform mainly includes two components: (1) an instrument that

can complete all steps of the chemiluminescence immunoassay automatically and (2) an integrated microfluidic chip which is

disposable and harmless. In the instrument, we adopt vacuum suction cups which are driven by linear motor to realize a simple,

effective and convenient control. The method of acridine esterification chemiluminescence is adopted to achieve a quantitative

detection, and a photomultiplier tube is used to detect photons from acridine ester producing in alkaline conditions. We use the

laser cutting machine and hot press machine to accomplish the product of microfluidic chips. The automated microfluidics-based

system is demonstrated by implementation of a chemiluminescence immunoassay for quantitative detection of ferritin. We

observe alinear relationship between CL intensity and the concentration of ferritin from 5.1 to 1300 ng mL

−1

and the limit of

detection (LoD) is 2.55 ng mL

−1

. At the same time, we also used the automated microfluidics-based system to test clinical serum

samples. The whole process of chemiluminescence experiment can complete within 45 min. We realize that this lab-on-a-chip

chemiluminescence immunoassay platform with features of automation and quantitation provides a promising strategy for POC

diagnosis.

Keywords Chemiluminescence .Microfluidicchip .Acridine ester .Ferritin

1 Introduction

The lab-on-a-chip (LoC) platforms have made significant

progress in diagnosis in the past years, due to its inherent

advantages such as reduced consumption of reagents, highly

integrated operation of the experiment, rapid detection of

multiple biomarkers (Gervais et al. 2011;Gorkinetal.2010;

Nahavandi et al. 2014;Ngetal.2010). There are some LoC

platforms which have been applied for a variety of biomarkers

including proteins, (Fan et al. 2008; Lee et al. 2013;Lietal.

2013; Liu et al. 2009,2011; McRae et al. 2016; Nie et al.

2014) nucleic acids, (Cai et al. 2014;Chenetal.2016,2017;

Fang et al. 2011; Gan et al. 2014; Liu et al. 2017; Zhang et al.

2011;Zhuangetal.2016) and cells (Hyun et al. 2015; Shen

et al. 2014) in blood or urine with the characteristics of the

high sensitivity and high throughput, but few of them can be

of the marketization.

The main challenges we face in LoC platforms are how to

transport the fluids in microchannels precisely (Li et al. 2014;

Song and Shum 2012) and design an automated instrument

which can sensitively detect the biochemical signal inside the

reaction reservoir and provide an quantitative result. Now

most drive using peristaltic pump make the vent hose directly

connect with the chip (or related links of pillar on chip), which

makes the chip production process more difficult and the ex-

periment operation more complicated. Centrifugal forces pro-

pel liquids radially outwards, which is considered a major

*Dongxu Zhang

zhangdongxu@xmu.edu.cn

*Shengxiang Ge

sxge@xmu.edu.cn

School of Information Science and Technology, Xiamen University,

Xiamen, Fujian, China

National Institute of Diagnostics and Vaccine Development in

Infectious Disease, Xiamen, Fujian, China

State Key Laboratory of Molecular Vaccinology and Molecular

Diagnostics, Xiamen University, Xiamen, Fujian, China

School of Public Health, Xiamen University, Xiamen, Fujian, China

Biomedical Microdevices (2018) 20: 91

https://doi.org/10.1007/s10544-018-0331-3

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Stepping forward in antibody-drug conjugate development

Article

Jun 2021
PHARMACOL THERAPEUT

Antibody-drug conjugates (ADCs) are cancer therapeutic agents comprised of an antibody, a linker and a small-molecule payload. ADCs use the specificity of the antibody to target the toxic payload to tumor cells. After intravenous administration, ADCs enter circulation, distribute to tumor tissues and bind to the tumor surface antigen. The antigen then undergoes endocytosis to internalize the ADC into tumor cells, where it is transported to lysosomes to release the payload. The released toxic payloads can induce apoptosis through DNA damage or microtubule inhibition and can kill surrounding cancer cells through the bystander effect. The first ADC drug was approved by the United States Food and Drug Administration (FDA) in 2000, but the following decade saw no new approved ADC drugs. From 2011 to 2018, four ADC drugs were approved, while in 2019 and 2020 five more ADCs entered the market. This demonstrates an increasing trend for the clinical development of ADCs. This review summarizes the recent clinical research, with a specific focus on how the in vivo processing of ADCs influences their design. We aim to provide comprehensive information about current ADCs to facilitate future development.

Green synthesized carbon quantum dots as chemiluminescence sensor for sulfanilamide detection

Article

Mar 2024
DYES PIGMENTS

Recent development of chemiluminescence for bioanalysis

Article

Sep 2023
TRAC-TREND ANAL CHEM

Optical Detection of Cancer Cells Using Lab-on-a-Chip

Article

Full-text available

Mar 2023

The global need for accurate and efficient cancer cell detection in biomedicine and clinical diagnosis has driven extensive research and technological development in the field. Precision, high-throughput, non-invasive separation, detection, and classification of individual cells are critical requirements for successful technology. Lab-on-a-chip devices offer enormous potential for solving biological and medical problems and have become a priority research area for microanalysis and manipulating cells. This paper reviews recent developments in the detection of cancer cells using the microfluidics-based lab-on-a-chip method, focusing on describing and explaining techniques that use optical phenomena and a plethora of probes for sensing, amplification, and immobilization. The paper describes how optics are applied in each experimental method, highlighting their advantages and disadvantages. The discussion includes a summary of current challenges and prospects for cancer diagnosis.

Merging microfluidics with luminescence immunoassays for urgent point-of-care diagnostics of COVID-19

Article

Nov 2022
TRAC-TREND ANAL CHEM

The Coronavirus disease 2019 (COVID-19) outbreak has urged the establishment of a global-wide rapid diagnostic system. Current widely-used tests for COVID-19 include nucleic acid assays, immunoassays, and radiological imaging. Immunoassays play an irreplaceable role in rapidly detecting COVID-19 and monitoring the patients for the assessment of their severity, risks of the immune storm, and prediction of treatment outcomes. Despite of the enormous needs for immunoassays, the widespread use of traditional immunoassay platforms is still limited by high cost and low automation, which are currently not suitable for point-of-care tests (POCTs). Microfluidic chips with the features of low consumption, high throughput, and integration, provide the potential to enable immunoassays for POCTs, especially in remote areas. Meanwhile, luminescence detection can be merged with immunoassays on microfluidic platforms for their good performance in quantification, sensitivity, and specificity. This review introduces both homogenous and heterogenous luminescence immunoassays with various microfluidic platforms. We also summarize the strengths and weaknesses of the categorized methods, highlighting their recent typical progress. Additionally, different microfluidic platforms are described for comparison. The latest advances in combining luminescence immunoassays with microfluidic platforms for POCTs of COVID-19 are further explained with antigens, antibodies, and related cytokines. Finally, challenges and future perspectives were discussed.

Intelligent hydrogels and their biomedical applications

Article

Full-text available

Sep 2022

Intelligent biomaterials can modify their properties in response to physical, chemical, and biological stimuli. These smart characteristics drive the innovation of biomaterials in therapy and diagnostics for detecting diseases and providing treatment at early stages. Mainly, hydrogels have gained significant interest in developing smart materials due to their excellent biocompatibility and ability to interact with body fluids that host condition-specific stimuli. Temperature, pressure, pH, light, ROS, cell metabolites, and other physicochemical factors specific to specific disease conditions were studied as major stimuli for designing intelligent biomaterials. The stimuli-responsive characteristic mainly depends on the sensitivity of the biomaterial to the stimuli factor and the tunable macromolecular structure of the materials. The method of biomaterial fabrication is critical in determining the physical and chemical properties of the biomaterial. Surface functionalisation, material blending, and crosslinking are commonly used to synthesise intelligent hydrogels to change the macromolecular structure. The impact and mechanism of these fabrication methods on the macromolecular structure and stimuli responsiveness of intelligent materials remain unidentified. This review focuses on strategies for transforming conventional hydrogels into intelligent hydrogels, their concerning mechanisms of stimuli-responsiveness and their biomedical applications.

Application of electrochemical biosensor based on metal organic framework materials in cancer detection

Article

Aug 2022

In recent years, there has been an increasing study of metal-organic frameworks (MOFs) materials because of their many excellent properties, such as stable skeleton structure, adjustable pore scale, and large specific surface area, which make MOFs a substrate for fixing bioprobes for building electrochemical biosensors. The electrochemical biosensor is a detection device that combines the high specificity of tumor markers with the high sensitivity of electrochemical sensors through sensitive elements, which has a major application in clinical cancer screening. This article provides an overview of the classification of metal-organic skeleton composites, summarizing the various markers (such as cancer markers, microRNAs, and DNA) used over the past five years to detect and predict their development as indicators of early cancer diagnosis.

A Review of Optical Imaging Technologies for Microfluidics

Article

Full-text available

Feb 2022

Microfluidics can precisely control and manipulate micro-scale fluids, and are also known as lab-on-a-chip or micro total analysis systems. Microfluidics have huge application potential in biology, chemistry, and medicine, among other fields. Coupled with a suitable detection system, the detection and analysis of small-volume and low-concentration samples can be completed. This paper reviews an optical imaging system combined with microfluidics, including bright-field microscopy, chemiluminescence imaging, spectrum-based microscopy imaging, and fluorescence-based microscopy imaging. At the end of the article, we summarize the advantages and disadvantages of each imaging technology.

Article

Jul 2021
CURR OPIN BIOTECH

Monitoring technologies for Process Analytical Technology (PAT) in mammalian cell cultures are often focusing on the same hand full parameters although a deeper knowledge and control of a larger panel of culture components would highly benefit process optimization, control and robustness. This short review highlights key advances in microfluidic affinity assays and microchip capillary electrophoresis (MCE). Aiming at the miniaturization and integration of PAT, these can detect at-line a variety of metabolites, proteins and Critical Quality Attributes (CQA’s) in a bioprocess. Furthermore, discrete analytical components, which can potentially support the translation of increasingly mature microfluidic technologies towards this novel application, are also presented as a comprehensive toolbox ranging from sample preparation to signal acquisition.

Siphon-Controlled Automation on a Lab-on-a-Disc Using Event-Triggered Dissolvable Film Valves

Article

Full-text available

Mar 2021

Within microfluidic technologies, the centrifugal microfluidic “Lab-on-a-Disc” (LoaD) platform offers great potential for use at the PoC and in low-resource settings due to its robustness and the ability to port and miniaturize ‘wet bench’ laboratory protocols. We present the combination of ‘event-triggered dissolvable film valves’ with a centrifugo-pneumatic siphon structure to enable control and timing, through changes in disc spin-speed, of the release and incubations of eight samples/reagents/wash buffers. Based on these microfluidic techniques, we integrated and automated a chemiluminescent immunoassay for detection of the CVD risk factor marker C-reactive protein displaying a limit of detection (LOD) of 44.87 ng mL−1 and limit of quantitation (LoQ) of 135.87 ng mL−1.

Centrifugal Micro-Channel Array Droplet Generation for Highly Parallel Digital PCR

Article

Full-text available

Dec 2016

Stable water-in-oil emulsion is essential to digital PCR and many other bioanalytical reactions that employ droplets as microreactors. We developed a novel technology to produce monodisperse emulsion droplets with high efficiency and high throughput using a bench-top centrifuge. Upon centrifugal spinning, the continuous aqueous phase is dispersed into monodisperse droplet jets in air through a micro-channel array (MiCA) and then submerged into oil as a stable emulsion. We performed dPCR reactions with a high dynamic range through the MiCA approach, and demonstrated that this cost-effective method not only eliminates the usage of complex microfluidic devices and control systems, but also greatly suppresses the loss of materials and cross-contamination. MiCA-enabled highly parallel emulsion generation combines both easiness and robustness of picoliter droplet production, and breaks the technical challenges by using conventional lab equipment and supplies.

A compact and integrated immunoassay with on-chip dispensing and magnetic particle handling

Article

Full-text available

Feb 2016
BIOMED MICRODEVICES

We present a compact diagnostic platform for a rapid and sensitive detection of plasma biomarkers. The platform consists of a disposable microfluidic polymer chip, a processing device including a lens-free and cost efficient sensor system and a setup for dispersion of magnetic particles. The biomarkers of interest are quantified by magnetic bead based immunoassays with chemiluminescent readout technology. With a novel system for dispersion and manipulation of the magnetic particles in combination with chemiluminescence detection, the sensitivity of the immunoassay is improved and enables a rapid assay in a microfluidic format. In the disposable chip, extra chambers for storage and dispensing of biomarker specific reagents are integrated, which reduce the need of external dosing devices and thereby the cost of the platform is decreased. Plasma biomarkers for monitoring of sepsis could be quantified at 10 pg/mL concentrations within a total time of 30 min by the present system. This contribution is a fundamental step towards the development of an automatic and compact Point-of-Care testing device for monitoring of patients at the intensive care unit.

A fully integrated and automated microsystem for rapid pharmacogenetic typing of multiple warfarin-related single-nucleotide polymorphisms

Article

Full-text available

Nov 2015

A fully integrated and automated microsystem consisting of low-cost, disposable plastic chips for DNA extraction and PCR amplification combined with a reusable glass capillary array electrophoresis chip in a modular-based format was successfully developed for warfarin pharmacogenetic testing. DNA extraction was performed by adopting a filter paper-based method, followed by "in situ" PCR that was carried out directly in the same reaction chamber of the chip without elution. PCR products were then co-injected with sizing standards into separation channels for detection using a novel injection electrode. The entire process was automatically conducted on a custom-made compact control and detection instrument. The limit of detection of the microsystem for the singleplex amplification of amelogenin was determined to be 0.625 ng of standard K562 DNA and 0.3 μL of human whole blood. A two-color multiplex allele-specific PCR assay for detecting the warfarin-related single-nucleotide polymorphisms (SNPs) 6853 (-1639G>A) and 6484 (1173C>T) in the VKORC1 gene and the *3 SNP (1075A>C) in the CYP2C9 gene was developed and used for validation studies. The fully automated genetic analysis was completed in two hours with a minimum requirement of 0.5 μL of input blood. Samples from patients with different genotypes were all accurately analyzed. In addition, both dried bloodstains and oral swabs were successfully processed by the microsystem with a simple modification to the DNA extraction and amplification chip. The successful development and operation of this microsystem establish the feasibility of rapid warfarin pharmacogenetic testing in routine clinical practice.

An Integrated Microfluidic Device Utilizing Dielectrophoresis and Multiplex Array PCR for Point-of-Care Detection of Pathogens

Article

Full-text available

Jul 2014
LAB CHIP

Early identification of the causative pathogens in physiological specimens that require no cultivation is essential for directing an evidence-based antimicrobial therapy in resource limited settings. Here, we describe an integrated microfluidic device for rapid identification of pathogens in complex physiological matrices such as blood. The device was designed and fabricated using SlipChip technologies, which integrated four channels processing independent samples and identifying up to twenty different pathogens. Briefly diluted whole human blood samples were directly loaded into the device for analysis. Pathogens were derived from blood by dielectrophoresis, retained in the array of grooves, and identified by multiplex array PCR in nanoliter volumes with end-point fluorescence detection. The universality of dielectrophoretic separation of pathogens from physiological fluids was evaluated with a panel of clinical isolates covering predominant bacterial and fungal species. Using this system, we simultaneously identified Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli O157:H7 within 3 hours. In addition to the prompt diagnosis of bloodstream infections, this method may be also utilized for differentiating microorganisms in other physiological and environmental samples.

Magnetic chemiluminescent immunoassay for human C-reactive protein on the centrifugal microfluidics platform

Article

Jan 2015

Droplet-based Multi-volume Digital PCR by Surface Assisted Multifactor Fluid Segmentation Approach

Article

Nov 2016

Here we developed the surface assisted multifactor fluid segmentation (SAMFS), an automated, fast and flexible approach for generating two dimensional droplet array with tunable droplet volumes, for multi-volume digital PCR. The SAMFS was developed based on the combination of robotic liquid handling and surface assisted droplet generation techniques, where a continuous aqueous stream flowed out from a capillary probe was segmented and immobilized on hydrophilic micropillars of a microchip into massive oil-covered droplets with the probe rapidly scanning over the microchip. We studied various factors affecting the droplet generation process, including micropillar top area, distance between adjacent micropillars, aqueous stream flow rate and microchip moving speed, and demonstrated a high droplet generation throughput up to 50 droplets/s and a largest droplet volume adjusting range from 0.25 nL to 350 nL. The SAMFS approach was adopted to form an oil-covered array of 994 droplets with four different volumes (1.2, 6, 30 and 150 nL droplets) required for multi-volume digital PCR within 8 min. The droplet array system was applied in absolute quantification of plasmid DNA under the multi-volume digital PCR mode with a dynamic range spanning four orders of magnitude, as well as measurement of HER2 expression levels in different breast cancer cell lines. The results are consistent to those obtained by quantitative real-time PCR method, while the present one has higher precision.

Spinning micropipette liquid emulsion generator for single cell whole genome amplification

Article

Oct 2016
LAB CHIP

Many on-chip approaches that use flow-focusing to pinch the continuous aqueous phase into droplets have become the most popular methods that provide monodisperse emulsion droplets. However, not every lab can easily adapt a microfluidic workflow into their familiar protocols. We develop an off-chip approach, spinning micro-pipette liquid emulsion (SiMPLE) generator, to produce highly stable monodisperse water-in-oil emulsions using a moving micropipette to disperse the aqueous phase in an oil-filled microcentrifuge tube. This method provides a simple way to produce picoliter-size droplets in situ with no dead volume during emulsification. With SiMPLE, single-cell emulsion whole genome amplification was performed to demonstrate that this novel method can seamlessly be integrated with experimental operations and supplies that most researchers are familiar with. The SiMPLE generator has effectively lowered the technical difficulties in applications relying on emulsion droplets.

Programmable Bio-nanochip Platform: A Point-of-Care Biosensor System with the Capacity To Learn

Article

Jul 2016
ACCOUNTS CHEM RES

The combination of point-of-care (POC) medical microdevices and machine learning has the potential transform the practice of medicine. In this area, scalable lab-on-a-chip (LOC) devices have many advantages over standard laboratory methods, including faster analysis, reduced cost, lower power consumption, and higher levels of integration and automation. Despite significant advances in LOC technologies over the years, several remaining obstacles are preventing clinical implementation and market penetration of these novel medical microdevices. Similarly, while machine learning has seen explosive growth in recent years and promises to shift the practice of medicine toward data-intensive and evidence-based decision making, its uptake has been hindered due to the lack of integration between clinical measurements and disease determinations. In this Account, we describe recent developments in the programmable bio-nanochip (p-BNC) system, a biosensor platform with the capacity for learning. The p-BNC is a "platform to digitize biology" in which small quantities of patient sample generate immunofluorescent signal on agarose bead sensors that is optically extracted and converted to antigen concentrations. The platform comprises disposable microfluidic cartridges, a portable analyzer, automated data analysis software, and intuitive mobile health interfaces. The single-use cartridges are fully integrated, self-contained microfluidic devices containing aqueous buffers conveniently embedded for POC use. A novel fluid delivery method was developed to provide accurate and repeatable flow rates via actuation of the cartridge's blister packs. A portable analyzer instrument was designed to integrate fluid delivery, optical detection, image analysis, and user interface, representing a universal system for acquiring, processing, and managing clinical data while overcoming many of the challenges facing the widespread clinical adoption of LOC technologies. We demonstrate the p-BNC's flexibility through the completion of multiplex assays within the single-use disposable cartridges for three clinical applications: prostate cancer, ovarian cancer, and acute myocardial infarction. Toward the goal of creating "sensors that learn", we have developed and describe here the Cardiac ScoreCard, a clinical decision support system for a spectrum of cardiovascular disease. The Cardiac ScoreCard approach comprises a comprehensive biomarker panel and risk factor information in a predictive model capable of assessing early risk and late-stage disease progression for heart attack and heart failure patients. These marker-driven tests have the potential to radically reduce costs, decrease wait times, and introduce new options for patients needing regular health monitoring. Further, these efforts demonstrate the clinical utility of fusing data from information-rich biomarkers and the Internet of Things (IoT) using predictive analytics to generate single-index assessments for wellness/illness status. By promoting disease prevention and personalized wellness management, tools of this nature have the potential to improve health care exponentially.

Magnetic chemiluminescent immunoassay for human C-reactive protein on the centrifugal microfluidics platform

Article

Jul 2015

Gregor F Czilwik

Human C-reactive protein (CRP) has been reported as the inflammatory biomarker with the highest reference for use in clinical practice. However, the existing analytical techniques are lacking the automation and simplicity, as desired for a prospective immunoassay format for point-of-care (PoC) analysis. We have developed an automated magnetic chemiluminescent immunoassay (MCIA) on a mobile analyser for rapid PoC determination of CRP. The MCIA is fully automated after the initial loading of sample and immunoreagents at the inlet ports. The automated protocol involves the transportation of magnetic capture microparticles between adjacent reaction compartments using a set of stationary magnets, a microfluidic polymer disposable and a specific centrifugal protocol. The developed MCIA has a sample-to-answer time of 25 min and hands-on time of approximately 5 min. It detects the entire pathophysiological range of CRP, as desired for clinically-relevant high sensitivity CRP immunoassay format, i.e. 3 - 81 ng mL-1 in diluted human serum with a limit of detection (LOD) and limit of quantification (LOQ) of 1.5 ng mL-1 and 1.8 ng mL-1, respectively.

A Filter Paper-Based Microdevice for Low-Cost, Rapid, and Automated DNA Extraction and Amplification from Diverse Sample Types

Article

Jul 2014
LAB CHIP

A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice is constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1-μL human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purifications from 0.25-1 μL human whole blood yielded 8.1-21.8 ng DNA, higher than those using QIAamp® DNA Micro kits. To realize DNA extractions from raw samples, an additional sample loading chamber containing a filter net with an 80-μm mesh size were designed in front of the extraction chamber for accommodating sample materials. Real-world samples, including whole blood, dried blood stains on Whatman 903® paper, dried blood stains on FTATM card, buccal swabs, saliva, and cigarette butts can all be processed in the system in 8 minutes. In addition, a multiplex amplification of 15 STR (short tandem repeat) loci and a Sanger-based DNA sequencing of the 520-bp GJB2 gene were accomplished from the filters that contain extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, “in-situ” PCR amplifications were successfully performed in the DNA extraction chamber following DNA purifications from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic-PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.