Paper-based Immunodevice Research Articles

Kirigami paper-based colorimetric immunosensor integrating smartphone readout for determination of humoral autoantibody immune response

Ultrasensitive, rapid, and low-cost paper-based immunodevice integrating a smartphone platform to detect ultra-low concentrations of tumour-specific anti-p53 autoantibodies (anti-p53aAbs) in human serum samples was developed. The immunodevice was fabricated based on the principles of kirigami (paper cutting and pasting art). It consisted of hydrophilic paper discs sandwiched between a superhydrophobic paper basement and polyethylene terephthalate-ethylene vinyl acetate (PET-EVA) laminate to delineate the sensing discs. The human p53 antigen (p53Ag) was covalently immobilized onto the pre-functionalized paper disc and used as the biorecognition element to capture anti-p53aAbs in human serum samples. The Fe3O4@SiO2-prNH2-Au@Pd0.30NPs-anti-IgG immunonanoprobes were used as the catalytic label for substrate (TMB/H2O2) oxidation, which generates blue colourimetric spots on the paper sensor. A smartphone installed on a custom-made imaging photo box equipped with colour analysis software was used as a reader to capture and quantitatively analyze the images. The immunodevice exhibited a linear increase in the colour pixel intensity with increasing concentrations of anti-p53aAbs from 10 pg.mL−1 to 25.0 ng.mL−1, with a sensitivity of 1.05 pixel.mL.pg−1 and a detection limit of 3.1 pg.mL−1. The fabricated proof-of-concept immunodevice could provide a simple, affordable, and minimally invasive method for cancer diagnosis at point-of-care.

Three-dimensional ring-oven washing technique for a paper-based immunodevice.

Washing is a standard step for enzyme-linked immunosorbent assays (ELISA) performed on a paper-based chip, in which nonspecific-binding antibodies and antigens should be removed completely from the paper surface. In this study, a novel three-dimensional (3D) washing strategy using a heating ring-oven was carried out on a paper-based chip. Compared with a plane washing mode by a ring-oven, this 3D washing strategy obtained a lower background, as gravity played an important role in the washing step. The paper-based chip was placed on a 3D plastic holder and the waste area was connected to a heating ring. Use of a heating waste area meant that the nonspecific-binding protein was continuously carried to the waste area through gravity and capillary action. The angle between the plastic holder and the ring plane was carefully selected. The effect of washing on different parts of the detection area was investigated by upconversion fluorescence and chemiluminescence (CL). This novel 3D washing strategy was performed for carcinoembryonic antigen detection through CL and a lower detection limit of 2 pg ml-1 was obtained. This approach provides an effective washing strategy to remove nonspecific-binding antibody from a paper-based immunodevice.

Paper-based fluorometric immunodevice with quantum-dot labeled antibodies for simultaneous detection of carcinoembryonic antigen and prostate specific antigen.

A method is described for simultaneous fluorometric determination of the biomarkers carcinoembryonic antigen (CEA) and prostate specific antigen (PSA) on the same zone of a paper-based immunodevice. Two kinds of CdTe quantum dots, with respective emission peaks at 525nm and 605nm under a single excitation wavelength of 272nm, were used to label the antibodies against CEA and PSA. Then the capture antibodies of CEA and PSA were immobilized on the same zone of the paper-based device. With the difference of the colors of the quantum dot fluorescence (green and orange), CEA and PSA can be detected on the same zone of the paper-based device. By using of sandwich immunoassay format, CEA and PSA can be simultaneously detected in human serum samples with a linear response in the 1.0-40ng·mL-1 concentration range for both. The recovery rates of the serum sample were in the range of 95-105%. The method has the potential of being applied to the simultaneous determination of various other kinds of substances on a single multichannel paper-based chip. Graphical abstract Schematic presentation of the simultaneous fluorometric detection of two cancer biomarkers on the same zone of the paper-based immunodevice is provided. Two kinds of CdTe quantum dots with different emission peaks under the same excitation wavelength are labeled on different detection antibodies.

An inkjet printing paper-based immunodevice for fluorescence determination of immunoglobulin G

A sequential inkjet printing strategy was utilized on a microfluidic paper-based fluorescence (FL) immunodevice for immunoglobulin G (IgG) detection.

Paper-based chemiluminescence immunodevice with temporal controls of reagent transport technique

A paper-based chemiluminescence (CL) immunodevice for detection of three biomarkers with the temporal controls of reagent transport technique was developed in this work. Sugar barriers were built on the paper-based chip for the controlled reagent transport technique to create programmable flow delays for the later CL detection. Different amount of sucrose which can manipulate the reagent migration rate was drawn precisely on the microchannels by a craft-cutting instrument. The paper-based immunodevice with three channels was fabricated by the cutting method. Also the localized incision protocol with one side adhesive tape and paper-folding method were adopted here to isolate the detection zone from flow channels. This simple origami step of the detection zone can eliminate possible reagent diffusion and flowing during antibody immobilization steps and numerous washings. By using the gold nanoparticles (AuNPs) as the carrier for loading multi horseradish peroxidase (HRP) labeled signal antibody, greatly enhanced sensitivity was achieved by HRP catalyzed luminol CL system. Under a sandwich immunoassay, carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA125) and carbohydrate antigen 199 (CA199) were detected on the paper-based chip. A limit of detection (LOD) of 0.03ng/mL, 0.2U/mL, 0.2U/mL was demonstrated, respectively. We believe that this paper-based CL immunodevice with controlled reagent transport technique can provide a new strategy of sensitive detection of multi-biomarkers in point-of-care (POC) diagnostics field.

Plasma treatment of paper for protein immobilization on paper-based chemiluminescence immunodevice

A novel protein immobilization method based on plasma treatment of paper on the low-cost paper-based immunodevice was established in this work. By using a benchtop plasma cleaner, the paper microzone was treated by oxygen plasma treatment for 4min and then the antibody can be directly immobilized on the paper surface. Aldehyde group was produced after the plasma treatment, which can be verified from the fourier transform infrared spectroscopy (FT-IR) spectra and x-ray photoelectron spectroscopy (XPS) spectra. By linked to aldehyde group, the antibody can be immobilized on the paper surface without any other pretreatment. A paper-based immunodevice was introduced here through this antibody immobilization method. With sandwich chemiluminescence (CL) immunoassay method, the paper-based immunodevice was successfully performed for carcinoembryonic antigen (CEA) detection in human serum with a linear range of 0.1–80.0ng/mL. The detection limit was 0.03ng/mL, which was 30 times lower than the clinical CEA level. Comparing to the other protein immobilization methods on paper-based device, this strategy was faster and simpler and had potential applications in point-of-care testing, public health and environmental monitoring.

Ring-Oven Washing Technique Integrated Paper-based Immunodevice for Sensitive Detection of Cancer Biomarker.

A paper-based microfluidic immunodevice has recently attracted considerable interest for point-of-care testing (POCT) and a washing procedure was used as a standard procedure in immunoassay to eliminate the nonspecific binding protein from a paper surface. However, the traditional washing method cannot get rid of the nonspecific binding protein more completely to get a lower background. In this work, a novel washing strategy with a ring-oven technique integrated on a paper-based immunodevice was presented, which can effectively wash a nonspecific binding protein and enable a low background for sensitive detection of the carcinoembryonic antigen (CEA). By immobilizing the antibody on the detection area and incorporating the temperature-controlled ring-oven under the paper-based device, the continuous washing solution can carry the nonspecific binding protein to the waste area freely by capillary force and then the waste area dried quickly by heating. The paper device, which is matched to the size of the ring-oven, is composed of eight microfluidic channels by the simple and rapid paper-cutting fabrication method. With the HRP-catalyzed 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 colorimetric detection method, a lower detection limit of 0.03 ng/mL CEA can be obtained by enzyme-linked immunosorbent assay (ELISA). The washing efficiency for the nonspecific binding protein was improved a lot compared to the traditional washing methods, and the established paper-based device can be used in the determination of CEA in human serum with high sensitivity. The paper-based device provides a new washing strategy for sensitive immunoassay and point-of-care diagnostics.

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 (+1.0V 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 0.001–10ngmL−1 with the detection limits down to 1.0 and 0.8pgmL−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.

A disposable immunosensor device for point-of-care test of tumor marker based on copper-mediated amplification

A paper-based immunodevice was fabricated for point-of-care test (POCT). The array device was simple and easily assembled. It comprised as many as 4×10 detection points on a single paper array. The immunosensor array was prepared by covalently immobilizing capture antibodies on corresponding working zone on a disposable paper array. With a sandwich-type immunoreaction, the CuO nanoparticles (CuO NPs)-labeled secondary antibody (Ab2) bioconjugates were captured in each working zone. The coordination of dithizone (DZ) at the surface of CdTe quantum dots (CdTe QDs) could strongly quench the green emission of CdTe QDs by a fluorescence resonance energy transfer (FRET) mechanism. After the Cu2+ was released from CuO NPs-Ab2, the fluorescence of CdTe QDs–DZ was “turn-on”. The fluorescence intensity would increase with the increasing of analytes. The calibration plot showed a good linear relationship between the fluorescence intensity and the logarithm value of the analytes concentration with the low detection limit. The immunosensor array was performed for cancer screening. The high throughput, low-cost, acceptable stability, reproducibility, sensitivity and accuracy showed good applicability of the proposed multiplex immunoassay in clinical diagnosis. The results indicated that the device could be applied to comprehensive sample and point-of-care detection.

3D Origami-based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device

A novel 3D microfluidic paper-based immunodevice, integrated with blood plasma separation from whole blood samples, automation of rinse steps, and multiplexed CL detections, was developed for the first time based on the principle of origami (denoted as origami-based device). This 3D origami-based device, comprised of one test pad surrounded by four folding tabs, could be patterned and fabricated by wax-printing on paper in bulk. In this work, a sandwich-type chemiluminescence (CL) immunoassay was introduced into this 3D origami-based immunodevice, which could separate the operational procedures into several steps including (i) folding pads above/below and (ii) addition of reagent/buffer under a specific sequence. The CL behavior, blood plasma separation, washing protocol, and incubation time were investigated in this work. The developed 3D origami-based CL immunodevice, combined with a typical luminuol-H(2)O(2) CL system and catalyzed by Ag nanoparticles, showed excellent analytical performance for the simultaneous detection of four tumor markers. The whole blood samples were assayed and the results obtained were in agreement with the reference values from the parallel single-analyte test. This paper-based microfluidic origami CL detection system provides a new strategy for a low-cost, sensitive, simultaneous multiplex immunoassay and point-of-care diagnostics.