Paper-based ELISA Research Articles

Rapid and accurate detection of Escherichia coli O157:H7 in beef using microfluidic wax-printed paper-based ELISA

Escherichia coli O157:H7 is a severe foodborne pathogen that causes lots of life-threatening diseases. In the search for a rapid, sensitive, portable and low-cost method to detect this pathogen, we developed a wax-printed paper-based enzyme-linked immunosorbent assay (P-ELISA) based on microfluidic paper-based analytical devices (μPADs), with the whole operation time being less than 3 h and only needing 5 μl samples for detection. The limit of detection (LOD) of E. coli O157:H7 reached 104 CFU ml-1, which is an order of magnitude higher than that of conventional ELISA (C-ELISA). The LOD in artificially contaminated beef samples is 1 CFU per 25 g after enriching the culture for 8 h. This method is superior to the molecular biology method in detection sensitivity and superior to C-ELISA and the national standard method in detection time and cost. Thus, the established P-ELISA method has good sensitivity, specificity and repeatability. It can be suitable for point-of-care testing without expensive and bulky instruments and can also provide a platform for detecting other pathogens, especially in areas that lack advanced clinical equipment.

Two Potential Clinical Applications of Origami-Based Paper Devices.

Detecting small amounts of analyte in clinical practice is challenging because of deficiencies in specimen sample availability and unsuitable sampling environments that prevent reliable sampling. Paper-based analytical devices (PADs) have successfully been used to detect ultralow amounts of analyte, and origami-based PADs (O-PADs) offer advantages that may boost the overall potential of PADs in general. In this study, we investigated two potential clinical applications for O-PADs. The first O-PAD we investigated was an origami-based enzyme-linked immunosorbent assay (ELISA) system designed to detect different concentrations of rabbit IgG. This device was designed with four wing structures, each of which acted as a reagent loading zone for pre-loading ELISA reagents, and a central test sample loading zone. Because this device has a low limit of detection (LOD), it may be suitable for detecting IgG levels in tears from patients with a suspected viral infection (such as herpes simplex virus (HSV)). The second O-PAD we investigated was designed to detect paraquat levels to determine potential poisoning. To use this device, we sequentially folded each of two separate reagent zones, one preloaded with NaOH and one preloaded with ascorbic acid (AA), over the central test zone, and added 8 µL of sample that then flowed through each reagent zone and onto the central test zone. The device was then unfolded to read the results on the test zone. The three folded layers of paper provided a moist environment not achievable with conventional paper-based ELISA. Both O-PADs were convenient to use because reagents were preloaded, and results could be observed and analyzed with image analysis software. O-PADs expand the testing capacity of simpler PADs while leveraging their characteristic advantages of convenience, cost, and ease of use, particularly for point-of-care diagnosis.

Atom transfer radical polymer-modified paper for improvement in protein fixation in paper-based ELISA

A newly modified paper-based enzyme-linked immunosorbent assay (P-ELISA) was established by immobilizing more proteins on the paper surface through an atom transfer radical polymerization (ATRP) reaction. In addition, introducing graphene oxide (GO) sheets, Au nanoparticles (AuNps) and two primary antibodies (Ab1s) led to signal amplification and cost reduction.

Ultrasensitive Paper-based ELISA by Introducing Atom Transfer Radical Polymer-modified Graphene Oxide Sheets and Gold Nanoparticles

In recent years, enzyme-linked immunosorbent assay (ELISA) plate fabricated on paper (P-ELISA) has been well developed. However, low sensitivity is its main disadvantage and has limited its use. In this study, a modified strategy was developed by introducing atom transfer radical polymer (ATRP)-modified graphene oxide (GO) sheets and gold nanoparticles (GNs). The results suggest the ATRP/GO/GNs-modified P-ELISA (AGGP-ELISA) achieves 1024 fold amplification and is suitable for rapid identification and qualitative detection, especially in small laboratories, or developing countries.

(Invited) Translating Paper-Based Diagnostic Devices into Clinically Relevant Applications

Developing methods to answer scientific questions is one of the practical approaches for doing science; optical microscopy, for example, has been a fundamental method of biological or medical discovery for more than three centuries. Building systems or tools to solve current issues existed in the world (e.g., infectious or chronic diseases) is also one of the attracting projects that we can involve and contribute. Due to this reason, for last few years, we have developed multiple “simple” technologies, which can be used in both developing and developed worlds. I will talk about what we are currently working on at National Tsing Hua University, including paper-based ELISA for the diagnosis of various diseases such as proliferative diabetic retinopathy, and other fiber-based diagnostic formats (i.e., cotton and bamboo), with their clinical validations, for clinically relevant diagnostic applications (septicemia and rapid diagnosis for organophosphorus and paraquat poisoning) in different divisions of medicine.

Development and Assessment of a Paper-based Enzyme-linked Immunosorbent Assay for the Colorimetric Diagnosis of Human Brucellosis

Brucellosis is one of the most common zoonotic diseases with widespread distribution. Traditional methods for diagnosis of brucellosis require long determination times with complex instruments that do not meet the needs of rapid, simple, and convenient analysis. Thus, a newly rapid and simple method for the colorimetric diagnosis of brucellosis was developed using a paper-based enzyme-linked immunosorbent assay (p-ELISA). A total of 135 serum samples were analyzed including 33 samples as healthy controls, 77 samples as the positive group with brucellosis, and 25 samples were infected with other bacteria. The major parameters were optimized for the reported protocol. To evaluate the method, receiver operating characteristic analysis and statistical analysis were carried out using these serum samples. In the established p-ELISA system, only 5 ml of serum were required to detect the antibodies of human brucellosis in suspected brucellosis patients in less than 2 hours. The receiver operating characteristic curve was plotted, and the area under the curve of receiver operating characteristic was 0.996 and shown to be a highly accurate diagnostic test. The sensitivity was determined to be 0.961, the specificity 0.983, and the cutoff value 2.260. The gray scale intensity of the human brucellosis patients group was significantly higher than for the healthy and the non-brucellosis patient groups (p 0.001). This newly developed system allows the rapid and easy diagnosis of human brucellosis.

Fast analysis of ketamine using a colorimetric immunosorbent assay on a paper-based analytical device

In this study, we designed a rapid colorimetric sensing system using competitive ELISA test on a microfluidic paper-based analytical device for the detection of ketamine, a frequently abused drug. Oral fluid was selected for the test to facilitate the collection of samples based on its advantages of low infection risk, noninvasiveness of sample collection, and decreased chance of sample adulteration. After optimization of the operation parameters, including reaction temperature, vibration washing time, reaction time, and antibody concentration, the resulting assay can be completed in as little as 6 min with a detection limit of 0.03 ng/mL. Moreover, we developed an Android smartphone app to analyze and measure the results of the test, further enhancing the test’s portability, and image recording and data transmission capabilities. In order to test the feasibility and performance of the optimized colorimetric assay, 90 oral fluid samples from drug abuse patients were tested. The paper-based platform features 90% sensitivity (confidence interval (CI): 76.34–97.21%) and 92% specificity (CI: 80.77–97.78%) for ketamine analysis. This competitive paper-based ELISA sensing system provides a rapid, convenient, sensitive, and high-throughput approach for drug monitoring.

Paper-based chemiluminescence enzyme-linked immunosorbent assay enhanced by biotin-streptavidin system for high-sensitivity C-reactive protein detection

High-sensitivity C-reactive protein (hs-CRP) has been regarded as a risk predictor of cardiovascular disease (CVD). Despite there are many methods to detect hs-CRP, quantitative, rapid, convenient, multiplex and highly sensitive measurement of it is still a challenge for point-of-care applications. In this study, we developed a paper-based ELISA to detect hs-CRP and the sensitive chemiluminescence was applied as detection signal. In this developed assay method, CRP concentration and chemiluminescence intensity were linearly correlated (r = 0.999) with a limit of detection (LOD) as low as 0.49 ng mL−1, which was comparable to that of conventional ELISA and superior to most of the current reported POCT methods for detection of hs-CRP. The precision of the assay was confirmed for low coefficient of variations, less than 7% for intra-assay and less than 10% for inter-assay. In clinical sample analysis, the results of hs-CRP detected by this assay were in good accordance with which obtained by commercial high sensitivity ELISA kit for in vitro diagnosis (r = 0.975). This assay required only 4 μL of sample and could be finished in less than 30 min. It may therefore be employed as a rapid pre-screening tool to identify patients with elevated risk of CVD.

Rapid detection of clenbuterol in milk using microfluidic paper-based ELISA

In this study, a paper-based microfluidic enzyme-linked immunosorbent assay (ELISA) was developed as a screening system for rapid detection of clenbuterol, which is illegally used as a growth promoter for food-producing animals. The microfluidic paper-based analytical device (μPAD) was combined with ELISA and the intrinsic properties of paper allowed the entrapment of antibody through cellulosic fibres, validating to be an alternative to 96-well ELISA microplate for food safety monitoring. Detection of clenbuterol in milk was achieved by measuring the intensity of colour change that was proportional to the analyte concentration with a detection limit of 0.2 ppb. The μPAD effectively reduces the cost, volume of reagents, and time required to run ELISA for food sample testing.

Improving paper-based ELISA performance through covalent immobilization of antibodies

Microfluidic paper-based analytical devices have been widely used for immunoassays, especially in resource-limited settings. Antibodies that have been immobilized on the cellulose surface by physical adsorption become unstable as a result of antibodies’ desorption caused by the washing process and lose their activity. In this paper, we present a new approach for the covalent immobilization of antibodies on the paper surface. More precisely, sodium periodate (NaIO4) was used to activate the cellulose for introducing aldehyde groups. Antibodies were bound to the oxidized cellulose paper surface through imine formation (Schiff base) between the aldehyde groups and the primary amine groups of the antibody. Thus, using α-fetoprotein (AFP) as a model, a new paper-based sandwich ELISA was developed. The linear range of the assay for AFP was from 0.1ng/mL to 11.2ng/mL, the detection limit was 0.04ng/mL, and the recovery rate of AFP spiked into human serum sample ranged from 89.2% to 101.5%. The intra- and inter-assay coefficients of variations obtained were 4.6–7.1% and 5.8–10.3% for standard AFP, respectively. Compared with the conventional paper-based ELISA, the developed assay exhibited superiority in terms of sensitivity, lower detection limit, stability and reliability. This simple and sensitive paper-based sandwich ELISA holds promise in clinical applications.

Zinc oxide nanorods functionalized paper for protein preconcentration in biodiagnostics

Distinguishing a specific biomarker from a biofluid sample containing a large variety of proteins often requires the selective preconcentration of that particular biomarker to a detectable level for analysis. Low-cost, paper-based device is an emerging opportunity in diagnostics. In the present study, we report a novel Zinc oxide nanorods functionalized paper platform for the preconcentration of Myoglobin, a cardiac biomarker. Zinc oxide nanorods were grown on a Whatman filter paper no. 1 via the standard hydrothermal route. The growth of Zinc oxide nanorods on paper was confirmed by a combination of techniques consisting of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS,) scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDX) analysis. The Zinc oxide nanorods modified Whatman filter paper (ZnO-NRs/WFP) was further tested for use as a protein preconcentrator. Paper-based ELISA was performed for determination of pre-concentration of cardiac marker protein Myoglobin using the new ZnO-NRs/WFP platform. The ZnO-NRs/WFP could efficiently capture the biomarker even from a very dilute solution (Myoglobin < 50 nM). Our ELISA results show a threefold enhancement in protein capture with ZnO-NRs/WFP compared to unmodified Whatman filter paper, allowing accurate protein analysis and showing the diagnostic concept.

Improved assessment of accuracy and performance indicators in paper-based ELISA

Paper-based devices are an excellent match for low-cost point-of-care testing (POCT) tools.

Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA

Intraocular vascular endothelial growth factor (VEGF) levels play an important role in the pathogenesis of blindness-related diseases, such as age-related macular degeneration (AMD). Here, we aimed to develop a paper-based enzyme-linked immunosorbent assay (P-ELISA) to analyze the suppression of aqueous VEGF concentrations following intravitreal injection (IVI) of anti-VEGF antibody (bevacizumab or ranibizumab). A total of 25 eyes with wet AMD, one with myopic neovascularization, and one with polypoidal choroidal vasculopathy were enrolled in this study. The limit of detection using P-ELISA was 0.03 pg/mL. Forty-six consecutive samples of aqueous humor were acquired. From all samples, 66.67% (10/15) achieved complete VEGF suppression (below the detection limit) within 5 weeks of receiving IVI of anti-VEGF antibody. Only 13.33% of samples (2/15) achieved complete VEGF suppression 5 weeks after receiving treatment. In some patients, elevated VEGF was still detected 5 weeks after receipt of anti-VEGF antibody, and all samples (10/10) were found to have elevated VEGF levels 49 days after treatment. Thus, we suggest that monthly IVI of anti-VEGF antibody may be required to ensure durable VEGF inhibition. Ultrasensitive P-ELISA can detect elevated VEGF at an earlier time point and may facilitate decision-making regarding appropriate treatment strategies.

Development of a Sampling Collection Device with Diagnostic Procedures.

Cervicovaginal fluid plays an important role in the detection of many female genital diseases, but the lack of suitable collection devices in the market severely challenges test success rate. Appropriate clinical sampling devices for cervicovaginal fluid collection would help physicians detect diseases and disease states more rapidly, efficiently, and accurately. The objective of this study was to develop a readily usable sampling collection device that would eliminate macromolecular interference and accurately provide specimens for further studies. This study was designed to develop an effective device to collect cervicovaginal fluid from women with symptoms of endometrial lesions, women appearing in the clinic for a routine Papanicolaou smear, and/or women seeking a routine gynecologic checkup. Paper-based assay, ELISA, and qNano were used to provide accurate diagnoses. A total of 103 patients successfully used the developed device to collect cervicovaginal fluid. Some of the collected specimens were used to detect glycogen, lactate, and pH for determining pathogen infection. Other specimen samples were tested for the presence of female genital cancer by comparing interleukin 6 concentration and microvesicle concentration. We proposed a noninvasive screening test for the diagnosis of female genital diseases using a dual-material collection device. The outer, nonwoven fabric portion of this device was designed to filter macromolecules, and the inner cotton portion was designed to absorb cervicovaginal fluid.

A straw-housed paper-based colorimetric antibody–antigen sensor

A simple and low cost paper-based ELISA sensor was developed by modifying the cellulose paper with chitosan and glutaraldehyde. The sensor incubation and wash was carried out using a straw for further convenience.

Paper-based ELISA to rapidly detect Escherichia coli

Escherichia coli is a generic indicator of fecal contamination, and certain serotypes cause food- and water-borne illness such as O157:H7. In the clinic, detection of bacteriuria, which is often due to E. coli, is critical before certain surgical procedures or in cases of nosocomial infection to prevent further adverse events such as postoperative infection or sepsis. In low- and middle-income countries, where insufficient equipment and facilities preclude modern methods of detection, a simple, low-cost diagnostic device to detect E. coli in water and in the clinic will have significant impact. We have developed a simple paper-based colorimetric platform to detect E. coli contamination in 5h. On this platform, the mean color intensity for samples with 105cells/mL is 0.118±0.002 (n=4), and 0.0145±0.003 (P<0.01⁎⁎) for uncontaminated samples. This technique is less time-consuming, easier to perform, and less expensive than conventional methods. Thus, paper-based ELISA is an innovative point-of-care diagnostic tool to rapidly detect E. coli, and possibly other pathogens when customized as appropriate, especially in areas that lack advanced clinical equipment.

Monitoring VEGF levels with low-volume sampling in major vision-threatening diseases: age-related macular degeneration and diabetic retinopathy.

The purpose of this article is to demonstrate the capacity of paper-based ELISA (P-ELISA) to monitor VEGF in patients requiring treatment for vision-threatening diseases. The most commonly encountered vision-threatening diseases are age-related macular degeneration (AMD) and diabetic retinopathy (DR), both of which may require short-term or life-long anti-VEGF injection treatment therapy. Accurate measurement of VEGF concentration in aqueous humor can provide significant and timely information to diagnose the disease state. Adequate and precise therapy may consequently be provided. At odds with conventional diagnostic approaches is the fact that a maximum of only 200 microliters of aqueous humor can be safely removed from the eye for testing. Fortunately, new diagnostic platforms, such as P-ELISA, require only minute volumes, i.e., approximately 2 microliters per test "well" and approximately 40 microliters total to quantify VEGF levels, and the testing process takes less than an hour. Thus, point-of-care (POC) diagnostics, such as P-ELISA, should be examined and improved upon as needed in order to develop an efficient tool for outpatient clinics and others to obtain semi-quantitative results that might facilitate accurate dosing of anti-VEGF treatment and delay or prevent the progression of AMD and DR.

Paper-Based ELISA for the Detection of Autoimmune Antibodies in Body Fluid—The Case of Bullous Pemphigoid

Bullous pemphigoid (BP), a common autoimmune blistering disease, is increasing in incidence and conveys a high mortality. Detection of autoantibodies targeting the noncollagenous 16A (NC16A) domain of type XVII collagen using enzyme-linked immunosorbent assay (ELISA) has demonstrated high sensitivity and specificity for diagnosing BP. We have developed a rapid, low-cost, and widely applicable ELISA-based system to detect the NC16A autoimmune antibody and then diagnose and monitor BP disease activity using a piece of filter paper, a wax-printer, and NC16A antigens. Both sera and/or blister fluids from 14 untreated BP patients were analyzed. The control group included healthy volunteers and patients with other blistering disorders such as pemphigus vulgaris. In our established paper-based ELISA (P-ELISA) system, only 2 μL of serum or blister fluid and 70 min were required to detect anti-NC16A autoimmune antibodies. The relative color intensity was significantly higher in the BP group than in the control groups when using either serum (P < 0.05) or blister fluid (P < 0.001) specimens from BP patients. The results of P- ELISA were moderately correlated with the titer of the commercial ELISA kit (MBL, Japan) (rho = 0.5680, P = 0.0011). This newly developed system allows for rapid and convenient diagnosis and/or monitoring of BP disease activity.

Monitoring the VEGF level in aqueous humor of patients with ophthalmologically relevant diseases via ultrahigh sensitive paper-based ELISA

The vascular endothelial growth factor (VEGF) level in aqueous humor has been used as an indicator to monitor specific diseases in the retinal ischemic condition. For clinical diagnosis, only about 200 μL of aqueous humor can be collected from the anterior chamber before the threat of anterior chamber collapse. It is necessary to develop an inexpensive diagnostic approach with the characteristics of highly sensitive, short operation duration, and requires small clinical sample quantities. To achieve the main objective of this study, we first prepared bevacizumab to be conjugated with HRP. We then deposited 2 μL aqueous humor from patients with different diseases onto each test zone of paper-based 96-well plates. After the colorimetric results were performed via ELISA protocol, the output signals were recorded using a commercial desktop scanner for analysis. In this study, only 2 μL from the aqueous humor of each patient was required for paper-based ELISA. The mean aqueous VEGF level was 14.4 pg/mL from thirteen patients (N = 13) with senile cataract as the control. However, the mean aqueous VEGF level from other patients with proliferative diabetic retinopathy (N = 14), age-related macular degeneration (N = 17), and retinal vein occlusion (N = 10) showed VEGF increases to 740.1 pg/mL, 383 pg/mL, and 219.4 pg/mL, respectively.

Cellulose‐Based Diagnostic Devices for Diagnosing Serotype‐2 Dengue Fever in Human Serum

Here, two types of cellulose-based in vitro diagnostic devices are demonstrated for the diagnosis of dengue virus infection in both buffer system and human serum: 1) paper-based ELISA for providing the semiquantitative information of the disease activity of serotype-2 dengue fever to healthcare persons (i.e., monitoring the disease activity with a specific serotype in single patients); 2) lateral flow immunoassays to screen for infection with serotype-2 dengue fever (i.e., rapid YES or NO diagnosis prepared for large populations, in terms of global public health). Paper-based ELISA (specific to serotype-2 dengue fever), which builds off of our previous studies and a revised previous ELISA procedure, owns multiple advantages: 1) high sensitivity (about 40 times higher than the current ELISA-based approaches, due to our therapeutic-based monoclonal antibody) and specificity (specific to dengue virus serotype-2 nonstructural protein-1 antigens); 2) tiny amount of sample and reagent used for single tests; 3) short operating duration (i.e., rapid diagnostic device); and, 4) inexpensiveness (appropriate for use in all developing and underdeveloped nations of the world). Due to the higher sensitivity and shorter operating duration of paper-based ELISA (compared with conventional ELISA, and lateral flow immunoassays also performed in this study), this study has not only been able to perform the diagnosis of dengue virus serotype-2 nonstructural protein-1 antigens in both buffer system and human serum but also to evaluate dengue virus serotype-2 envelope proteins in the buffer system, thus successfully achieving the first such use of these proteins as the target antigen for the development of diagnostic tools. These results provide a more comprehensive understanding for the genesis of dengue fever diagnostic tools (through antibody-antigen recognition).