Paper-based electrochemiluminescent 3D immunodevice for lab-on-paper, specific, and sensitive point-of-care testing

Recent research on microfluidic paper-based analytical devices (μPADs) has shown that paper has great potential for the fabrication of low-cost diagnostic devices for healthcare and environmental monitoring applications. Herein, electrochemiluminescence (ECL) was introduced for the first time into μPADs that were based on screen-printed paper-electrodes. To further perform high-specificity, high-performance, and high-sensitivity ECL on μPADs for point-of-care testing (POCT), ECL immunoassay capabilities were introduced into a wax-patterned 3D paper-based ECL device, which was characterized by SEM, contact-angle measurement, and electrochemical impedance spectroscopy. With the aid of a home-made device-holder, the ECL reaction was triggered at room temperature. By using a typical tris(bipyridine)ruthenium-tri-n-propylamine ECL system, this paper-based ECL 3D immunodevice was applied to the diagnosis of carcinoembryonic antigens in real clinical serum samples. This contribution further expands the number of sensitive and specific detection modes of μPADs.     

Battery-triggered ultrasensitive electrochemiluminescence detection on microfluidic paper-based immunodevice based on dual-signal amplification strategy

Dual-signal amplification strategy for ultrasensitive electrochemiluminescence (ECL) multiplexed immunoassay on microfluidic paper-based analytical devices (μ-PADs) was demonstrated. This dual-signal amplification technique was achieved by employing graphene oxide-chitosan/gold nanoparticles (GCA) immunosensing platform and [4,4′-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid] (P-acid) functionalized nanoporous silver (P-acid/NPS) signal amplification label. For further low-cost and disposable applications, battery-triggered constant-potential ECL (+1.0 V for P-acid label (vs. Ag/AgCl auxiliary electrode)) was applied on this paper-based immunodevice with the aid of a home-made voltage-tunable power device, allowing the traditional electrochemical workstation to be abandoned. We found that two tumor markers could be sequentially detected in the linear ranges of 0.003–20 and 0.001–10 ng mL⁻¹ with the detection limits down to 1.0 and 0.8 pg mL⁻¹, respectively, by simply reversing the connection mode on two working electrodes. The results exhibited excellent precision and high sensitivity of such immunoassay, and it also demonstrated that this battery-triggered ECL paper-based immunodevice could provide a rapid, simple and simultaneous multiplex immunoassay with high throughput, low-cost and low detection limits for point-of-care testing.     

Blood separation on microfluidic paper-based analytical devices

A microfluidic paper-based analytical device (μPAD) for the separation of blood plasma from whole blood is described. The device can separate plasma from whole blood and quantify plasma proteins in a single step. The μPAD was fabricated using the wax dipping method, and the final device was composed of a blood separation membrane combined with patterned Whatman No.1 paper. Blood separation membranes, LF1, MF1, VF1 and VF2 were tested for blood separation on the μPAD. The LF1 membrane was found to be the most suitable for blood separations when fabricating the μPAD by wax dipping. For blood separation, the blood cells (both red and white) were trapped on blood separation membrane allowing pure plasma to flow to the detection zone by capillary force. The LF1-μPAD was shown to be functional with human whole blood of 24-55% hematocrit without dilution, and effectively separated blood cells from plasma within 2 min when blood volumes of between 15-22 μL were added to the device. Microscopy was used to confirm that the device isolated plasma with high purity with no blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was 2.62% (n = 10) and 5.84% (n = 30) for within-day and between day precision, respectively. Our proposed blood separation on μPAD has the potential for reducing turnaround time, sample volume, sample preparation and detection processes for clinical diagnosis and point-of care testing.     

Electrochemical immunoassay on a 3D microfluidic paper-based device

A high-throughput, simple, fast, low-cost and sensitive paper-based electrochemical immunodevice has been demonstrated based on a functionalized 3D paper-based device for point-of-care diagnosis.     

Rechargeable battery-triggered electrochemiluminescence detection on microfluidic origami immunodevice based on two electrodes

A low-cost, portable rechargeable battery was firstly used to fabricate a battery-triggered, screen-printed two-electrode electrochemiluminescent immunoassay on a 3D microfluidic origami electronic device.     

Paper‐Based Electrochemiluminescent 3D Immunodevice for Lab‐on‐Paper,Specific, and Sensitive Point‐of‐Care Testing

Recent research on microfluidic paper‐based analytical devices (μPADs) has shown that paper has great potential for the fabrication of low‐cost diagnostic devices for healthcare and environmental monitoring applications. Herein, electrochemiluminescence (ECL) was introduced for the first time into μPADs that were based on screen‐printed paper‐electrodes. To further perform high‐specificity, high‐performance, and high‐sensitivity ECL on μPADs for point‐of‐care testing (POCT), ECL immunoassay capabilities were introduced into a wax‐patterned 3D paper‐based ECL device, which was characterized by SEM, contact‐angle measurement, and electrochemical impedance spectroscopy. With the aid of a home‐made device‐holder, the ECL reaction was triggered at room temperature. By using a typical tris(bipyridine)ruthenium–tri‐n‐propylamine ECL system, this paper‐based ECL 3D immunodevice was applied to the diagnosis of carcinoembryonic antigens in real clinical serum samples. This contribution further expands the number of sensitive and specific detection modes of μPADs.     

A lab-on-a-chip for rapid blood separation and quantification of hematocrit and serum analytes

In this work, a new lab-on-a-chip for rapid analysis of low volume blood samples was designed, fabricated and demonstrated for integration of serum separation, hematocrit evaluation, and protein quantitation. Blood separation was achieved using microchannel flow-based separation. A novel method for evaluating hematocrit from microfluidic flow-separated blood samples was developed using gray scale analysis of a point-and-shoot digital photograph of separated blood in a micochannel. Protein quantitation was subsequently performed in a high surface area-to-volume ratio microfluidic chemiluminescent immunoassay using cell depleted serum produced by microfluidic flow-based separation of whole blood samples. All three steps were achieved in a single microchannel with separation of blood samples and hematocrit evaluation in less than 1 min, and protein quantitation in 5 min.     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

Three-dimensional paper microfluidic devices assembled using the principles of origami

We report a method, based on the principles of origami (paper folding), for fabricating three-dimensional (3-D) paper microfluidic devices. The entire 3-D device is fabricated on a single sheet of flat paper in a single photolithographic step. It is assembled by simply folding the paper by hand. Following analysis, the device can be unfolded to reveal each layer. The applicability of the device to chemical analysis is demonstrated by colorimetric and fluorescence assays using multilayer microfluidic networks.     

Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.     

A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami

The lab-on-a-chip concept has led to several point-of-care (POC) diagnostic microfluidic platforms. However, few of these can process raw samples for molecular diagnosis and fewer yet are suited for use in a resource-limited setting without permanent electrical infrastructure. We present here a very low cost paper microfluidic device for POC extraction of bacterial DNA from raw viscous samples--a challenge for conventional microfluidic platforms. This is an example of "microfluidic origami" in that the system is activated by folding; demonstrated here is room temperature cell lysis and DNA extraction from pig mucin (simulating sputum) spiked with E. coli without the use of external power. The microfluidic origami device features dry reagent storage and rehydration of the lysis buffer. We demonstrate DNA extraction from samples with a bacterial load as low as 33 CFU ml(-1). Extraction times, starting from the raw sample, have been optimized to about 1.5 h without the use of external power, or to within 1 h using an oven or a heater block. The fabrication of this paper microfluidic device can be translated into high volume production in the developing world without the need for a semiconductor clean room or a microfabrication facility. The sample preparation can be performed with the addition of just the sample, water, ethanol and elute buffer to the device, thus reducing chemical hazards during transport and handling.     

Ultrahigh throughput beehive-like device for blood plasma separation

We report here a low-cost, rapid-prototyping, and beehive-like multilayer polymer microfluidic device for ultrahigh-throughput blood plasma separation. To understand the device physics and optimize the device structure, the effect of cross-sectional dimension and operational parameter on particle focusing behavior was explored using a single spiral microchannel device. Then, the blood plasma separation performance of the determined channel structure was validated using the blood samples with different hematocrits (HCTs). It was found that a high separation efficiency of 99% could be achieved using the blood sample with an HCT of 0.5% at a high throughput of 1 mL/min. Finally, a multilayer microfluidic device with a novel beehive-like multiplexing channel arrangement was developed for ultrahigh-throughput blood plasma separation. The prototype device could be fabricated within ∼1 hour utilizing the laser cutting and thermal lamination methods. The total processing throughput could reach up to 72 mL/min for 0.5% HCT sample with a plasma separation ratio close to 90%. Our device may hold potentials for the ultrahigh-throughput separation of blood plasma from large volume blood samples for downstream disease diagnosis.     

Lab-on-paper-based devices using chemiluminescence and electrogenerated chemiluminescence detection

As an analytical support, paper, being low cost, highly abundant, of high porosity, disposable or biodegradable, and easy to use, store, transport, and print, has excellent chemical compatibility with many applications. Since the first microfluidic paper-based analytical device (μ-PAD or lab-on-paper) was proposed, the paper-based assay has never attracted as much attention as it does now. There has recently been rapidly increasing interest in using sensitive luminescence methods, for example chemiluminescence (CL) and electrogenerated chemiluminescence (ECL), as the detection strategy for lab-on-paper devices. Because of their intrinsic characteristics, CL and ECL provide outstanding performance while retaining the simplicity, low cost, multifunctionality, versatility, flexibility, and disposability of μ-PADs. The objective of this review is to cover the development of lab-on-paper-based devices using CL and ECL detection, including fabrication of paper devices, construction of sensing interfaces, signal amplification strategies, external instruments used, and applications. We believe that lab-on-paper devices with CL and ECL detection methods will meet the diverse requirements of point-of-care diagnosis.     

Paper‐Origami‐Based Multiplexed Malaria Diagnostics from Whole Blood

We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.     

Paper-based three-dimensional microfluidic device for monitoring of heavy metals with a camera cell phone

A 3D paper-based microfluidic device has been developed for colorimetric determination of selected heavy metals in water samples by stacking layers of wax patterned paper and double-sided adhesive tape. It has the capability of wicking fluids and distributing microliter volumes of samples from single inlet into affrays of detection zones without external pumps, thus a range of metal assays can be simply and inexpensively performed. We demonstrate a prototype of four sample inlets for up to four heavy metal assays each, with detection limits as follows: Cu (II)?=?0.29 ppm, Ni(II)?=?0.33 ppm, Cd (II)?=?0.19 ppm, and Cr (VI)?=?0.35 ppm, which provided quantitative data that were in agreement with values gained from atomic absorption. It has the ability to identify these four metals in mixtures and is immune to interferences from either nontoxic metal ions such as Na(I) and K(I) or components found in reservoir or beach water. With the incorporation of a portable detector, a camera mobile phone, this 3D paper-based microfluidic device should be useful as a simple, rapid, and on-site screening approach of heavy metals in aquatic environments.

A microfluidic device for continuous, real time blood plasma separation

A microfluidic device for continuous, real time blood plasma separation is introduced. The principle of the blood plasma separation from blood cells is supported by the Zweifach-Fung effect and was experimentally demonstrated using simple microchannels. The blood plasma separation device is composed of a blood inlet, a bifurcating region which leads to a purified plasma outlet, and a concentrated blood cell outlet. It was designed to separate blood plasma from an initial blood sample of up to 45% inlet hematocrit (volume percentage of cells). The microfluidic network was designed using an analogous electrical circuit, as well as analytical and numerical studies. The functionality of this device was demonstrated using defibrinated sheep blood. During 30 minutes of continuous blood infusion through the device, all the erythrocytes (red blood cells) traveled through the device toward the concentrated blood outlet while only the plasma was separated at the bifurcating regions and flowed towards the plasma outlet. The device has been operated continuously without any clogging or hemolysis of cells. The experimentally determined plasma selectivity with respect to blood hematocrit level was almost 100% regardless of the inlet hematocrit. The total plasma separation volume percent varied from 15% to 25% with increasing inlet hematocrit. Due to the device's simple structure and control mechanism, this microdevice is expected to be used for highly efficient continuous, real time cell-free blood plasma separation from blood samples for use in lab on a chip applications.     

Electrochemical detection of glucose from whole blood using paper-based microfluidic devices

Electrochemical paper-based analytical devices (ePADs) with integrated plasma isolation for determination of glucose from whole blood samples have been developed. A dumbbell shaped ePAD containing two blood separation zones (VF2 membranes) with a middle detection zone was fabricated using the wax dipping method. The dumbbell shaped device was designed to separate plasma while generating homogeneous flow to the middle detection zone of the ePAD. The proposed ePADs work with whole blood samples with 24–60% hematocrit without dilution, and the plasma was completely separated within 4 min. Glucose in isolated plasma separated was detected using glucose oxidase immobilized on the middle of the paper device. The hydrogen peroxide generated from the reaction between glucose and the enzyme pass through to a Prussian blue modified screen printed electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal detection potential for H₂O₂ (−0.1 V versus Ag/AgCl reference electrode) were proportional to glucose concentrations in the whole blood. The linear range for glucose assay was in the range 0–33.1 mM (r² = 0.987). The coefficients of variation (CVs) of currents were 6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration at 3.4, 6.3, and 15.6 mM, respectively. Because each sample displayed intra-individual variation of electrochemical signal, glucose assay in whole blood samples were measured using the standard addition method. Results demonstrate that the ePAD glucose assay was not significantly different from the spectrophotometric method (p = 0.376, paired sample t-test, n = 10).     

Dielectrophoretic separation of platelets from diluted whole blood in microfluidic channels

The dielectrophoresis (DEP) phenomenon is used to separate platelets directly from diluted whole blood in microfluidic channels. By exploiting the fact that platelets are the smallest cell type in blood, we utilize the DEP-activated cell sorter (DACS) device to perform size-based fractionation of blood samples and continuously enrich the platelets in a label-free manner. Cytometry analysis revealed that a single pass through the two-stage DACS device yields a high purity of platelets (approximately 95%) at a throughput of approximately 2.2 x 10(4) cells/second/microchannel with minimal platelet activation. This work demonstrates gentle and label-free dielectrophoretic separation of delicate cells from complex samples and such a separation approach may open a path toward continuous screening of blood products by integrated microfluidic devices.     

Simple and covalent fabrication of a paper device and its application in sensitive chemiluminescence immunoassay

In this paper, chemiluminescence immunoassay (CLIA) was introduced into the recently proposed microfluidic paper-based analytical devices (μPADs) through covalent fabrication strategy for the first time. This novel paper-based CLIA, with high-throughput, rapid, stable and reusable CL response to trace amounts of analyte in real biological samples, combines the simplicity and low-cost of the μPADs with the high sensitivity and selectivity of CLIA. Periodate oxidation, which can form covalent bonds between polysaccharides and proteins, was used for activation of μPADs to covalently immobilize antibodies on μPADs in this work for the first time. Thus, the paper-based sandwich CLIA and regeneration of it can be easily realized for further development of this technique in sensitive, specific and low-cost applications. The application test of this paper-based CLIA was successfully performed, as a model, through the determination of biomarkers in human serum on paper microzone plate. The paper-based CLIA will be very useful when the level of an analyte in real biological sample is important for point-of-care testing, public health and environmental monitoring in remote regions, developing or developed countries.     

Fully integrated lab-on-a-disc for simultaneous analysis of biochemistry and immunoassay from whole blood

We report a fully integrated device that can perform both multiple biochemical analysis and sandwich type immunoassay simultaneously on a disc. The whole blood is applied directly to the disposable "lab-on-a-disc" containing different kinds of freeze-dried reagents for the blood chemistry analysis as well as reagents required for the immunoassay. The concentrations of different kinds of analytes are reported within 22 min by simply inserting a disc to a portable device. Using the innovative laser irradiated ferrowax microvalves together with the centrifugal microfluidics, the total process of plasma separation, metering, mixing, incubation, washing, and detection is fully automated. The analyzer is equipped with an optical detection module to measure absorbances at 10 different wavelengths to accommodate the various kinds of reaction protocols. Compared to the conventional blood analysis done in clinical laboratories, it is advantageous for point-of-care applications because it requires a smaller amount of blood (350 μL vs. 3 mL), takes less time (22 min vs. several days), does not require specially trained operators or expensive instruments to run biochemical analysis and immunoassay separately.