Sample Pad Research Articles

Rapid and visual detection of benzothiostrobin residue in strawberry using quantum dot-based lateral flow test strip

A quantitative method was developed for the rapid, visual detection of benzothiostrobin residue in strawberry using the conjugates of CdSe/ZnS (core/shell) quantum dots (QDs) and anti-benzothiostrobin antibody (IgG) based fluorescence lateral flow test strip (FLFTS). The IgG with a binding specificity to the target antigen benzothiostrobin-BSA (a conjugate of benzothiostrobin and bovine serum albumin) was labeled with the QDs to prepare the QD-IgG bioprobes. The primary strategy included the designation of three test lines (T lines), which were sprayed with different concentrations of the antigen. After the spiked samples were added to the sample pad for 15 min at 37 °C, the results were visualized with the unaided eye with using ultraviolet (UV) protective goggles under UV lamp with a detection limit of 25 μg/L. Moreover, the visual detection method together with a gel imager, could be used as a quantitative method. The cut-off values for the T line 1, 2, and 3 were 1200, 600, and 150 μg/L, respectively. So multi-concentration and visual detection of spiked samples had been achieved. At last, the detection results of the spiked samples by FLFTS were consistent with the results of the UPLC-MS/MS method.

Vertical flow-based paper immunosensor for rapid electrochemical and colorimetric detection of influenza virus using a different pore size sample pad

We present a novel vertical flow-based paper immunosensor for the rapid and sensitive electrochemical and colorimetric detection of influenza H1N1 viruses using a different pore size (DP) sample pad. The DP sample pad consisted of two different pore size papers: larger pores (diameter: 11 µm) facing the inlet, and smaller pores (diameter: 0.45 µm) facing the conjugate pad. This sample pad offered moderate and uniform flows, and hence concentrated horseradish peroxidase tagged antibodies (HRP-Abs)-H1N1 complexes from 40 µL of sample volumes on a conjugate pad for 2 min after sample injection, thereby providing fast detection (6 min for both detection methods) with 100 µL of flushing afterwards, high sensitivity, and the simplicity of the sensor. The filtration characteristics of the DP sample pad were evaluated using fluorescent beads, indicating that only small-sized bio-particles such as viruses can pass through the sample pad. Sandwich immunoreactions of HRP-Ab-H1N1-Ab were performed on the gold paper electrode of the immunoStrip, which was determined by electrochemical impedance spectroscopy (EIS) measurements. Simultaneously, the color signal of free HRP-Ab captured on the colorimetric zone was obtained using a scanner, and the intensity was analyzed using ImageJ. This immunosensor detected H1N1 virus concentration as low as 3.3 plaque forming units (PFU)/mL (phosphate buffer saline; PBS) and 4.7 PFU/mL (saliva) by EIS, and 1.34 PFU/mL (PBS) and 2.27 PFU/mL (saliva) by the colorimetric method. Furthermore, integrating these two detection methods can reduce false results with double assurance, and this device can provide a simple and economical on-site detection platform.

Development of Lateral Flow Immunochromatographic Strips for Micropollutants Screening Using Colorants of Aptamer Functionalized Nanogold Particles Part I Methodology and Optimization.

A methodology of lateral flow immunochromatographic strip based on aptamer was developed for on-site detection of the small molecule micropollutants. In the present study, we try for the first time to investigate the feasibility of developing a strip assay for the analysis of micropollutants as methodological prototypes by combining the high selectivity and affinity of aptamers with the unique optical properties of nanogolds. This quantitative method was based on the competition for the aptamer between targets and DNA probes. Crucial parameters that might influence the sensitivity, such as the size of nanogolds, amount of aptamer, type and pH of streptavidin, type of nitrocellulose (NC) membrane, blocking procedure, and reading time, were systematically investigated to obtain the optimum assay performance. With the optimized conditions [nanogolds 25 nm, 50 μM aptamer, pH 8 of GSA (a type of streptavidin named "SA Gold," which is a sulfhydrylization streptavidin), Millipore HFC 135 NC membrane, 1% bovine serum albumin as the blocking agent and added in the running buffer and sample pad soakage agents, and 20 min reading time] the aptamer-based lateral flow assay will show a low visual limit of detection and scanning reader LOD. The strip for on-site screening using colorants of aptamer functionalized nanogold particles did not require any complicated equipment and was a potential portable tool for rapid identification of micropollutants.

A lateral flow immunochromatographic strip test for rapid detection of hexoestrol in fish samples.

A lateral flow immunochromatographic strip test was developed for rapid and sensitive on-site detection of hexoestrol (HES) residues in fish samples with colloidal gold labelling of the anti-HES monoclonal antibody. The strip is composed of a sample pad, a conjugate reagent pad, an absorbent pad and a test membrane containing a control line and a test line. The sensitivity (half inhibitory concentration, IC50) of the strip in the detection of fish extract samples was confirmed to be 1.86 µg kg−1, and the limit of detection value was 0.62 µg kg−1. For intra-assay and inter-assay reproducibility, recoveries of HES-spiked samples ranged from 86.3% to 92.3% and 85.8% to 93.4%, coefficients of variation were 2.91–4.64% and 4.24–5.17%, respectively. High-performance liquid chromatography was employed to confirm the performance of the strip. The strip test takes less than 10 min, and thus provides a repaid method for on-site detection of HES residues.

AERO: An Objective Peripheral Edema Measurement Device.

Peripheral edema is of ten assessed by medical prof essionals to gain insights about development of many patient conditions. Currently, doctors assess edema by pushing on the swollen area of a patient. The difficulty with this method is the inconsistency assessment results between different physicians due to the subjective nature of the practice. The proposed solution uses AERO (or Air Edema RepOrting), a mobile, handheld device consisting of a high-speed camera with a macro lens, LED light, and compressed air to create edema indentation and capture images of the skin /tissue response. A MATLAB program analyzes these images and determines the correct level of edema. AERO was tested with a LifeformOR pitting edema trainer (four pieces of skin sample that represent the four edema levels). Various system configurations, such as the air pressure, camera to sample distance, and air incidence angle, were examined and optimal parameters were identified. Results demonstrated that AERO was able to distinguish the four levels of edema sample pads. This solution can potentially provide a quantified, standard evaluation of peripheral edema for both home and health care environments.

A lateral flow strip based on gold nanoparticles to detect 6-monoacetylmorphine in oral fluid.

We used lateral flow strips based on gold nanoparticles to detect 6-monoacetylmorphine (6-MAM; heroin's unique metabolite) in oral fluid samples. In this competitive lateral chromatographic immunoassay, the 6-MAM was chemically synthesized and conjugated to bovine serum albumin. The results were qualitatively detected via the colour change of the test line. By using a proper sample pad, a suitable nitrocellulose membrane and a customized sponge device adsorbed the oral fluid directly from the mouth; the total test time was 3 min. The sensitivity of the assay was 4.0 ng ml−1 without any cross-reactivity with 10 normal drugs, which are widely subject to abuse, including morphine and codeine. This test could be easily used on site to detect heroin in oral fluid, and it could be a promising product in the future including for driving under the influence.

Quantitative and rapid detection of C-reactive protein using quantum dot-based lateral flow test strip

A novel QD-based immunoassay on a paper-based lateral flow system has been developed to quantitatively detect C-reactive protein (CRP). Different standard CRP antigens from 1 to 200 μg mL−1 were diluted 200-fold and only 60 μL diluted sample were needed to load onto the sample pad. The QD fluorescence signals on the test line and the control line were able to be observed within 3 min after the initiation of assay, and the limit of detection was as sensitive as 0.30 ng mL−1 by measuring the fluorescence intensity immediately afterwards with fluorescence immunoassay analyzer. The linearity on the detection of QD fluorescence signals has been established well in the range of 0.5 ng mL−1 and 1 μg mL−1 for CRP. The precision of the assay has been confirmed for low coefficient of variation (CV), satisfying less than 15% (intra-assay and inter-assay), and the accuracy of assay meets the requirements with the mean recovery of the control was 102.63%. These results indicated that such newly developed platform was reliable with high sensitivity, rapidness, and could cover a broad range of target concentrations. Furthermore, a total of 135 human serum clinical samples with inflammation or infection with the concentration of CRP from 0.2 to 200 μg mL−1 has been used to check the performance of this QD-based LFIA, it correlated very well with Roche Tina-quant CRP (Latex) (r = 0.966, n = 135).

Wax-Assisted One-Step Enzyme-Linked Immunosorbent Assay on Lateral Flow Test Devices.

Lateral flow tests (LFTs) are widely used analytical tools characterized by portability, operator simplicity and short analysis times. A remaining challenge is their limited analytical sensitivity, which in classical immunoassay formats is overcome by enzyme-linked immunosorbent assay (ELISA) formats. The implementation of ELISA to an LFT format however, is hampered by the complexity of the procedure requiring the enzyme substrate addition after sample addition. In this work, a simple method for automation of this procedure without user interference is presented. Originally used sample pads of LFTs have been replaced by hydrophobic wax-modified filter paper-based sample pads to realize a delayed flow a pre-deposited colorimetric ELISA substrate without other alterations to the classical lateral-flow immunoassay format. The performance of the system has been characterized by visualizing flow behavior and final proof-of-concept is provided by a model mouse IgG assay, achieving a limit of detection of 15.8 ng mL-1 from just a single application of the sample solution.

Engineering a simple lateral flow device for animal blood coagulation monitoring.

Increasing numbers of animals are diagnosed with thromboembolism, requiring anticoagulation treatment to prevent thrombotic events. Frequent and periodic coagulation monitoring is critical to ensure treatment effectiveness and patient safety by limiting blood coagulation ability within the desired therapeutic range. Point-of-care diagnostics is an ideal candidate for frequent coagulation monitoring due to rapid test results and no need for laboratory setting. This article reports the first utilization of no-reaction lateral flow assay (nrLFA) device for simple and low-cost animal blood coagulation monitoring in resource-limited setting. The nrLFA device consists of sample pad, analytical membrane and wicking pad, without conjugate pad, reagent printing or membrane drying. Citrated and heparinized animal blood were utilized to mimic different blood coagulation abilities in vitro by adding reversal agents CaCl2 and protamine sulfate. The travel distance of red blood cells (RBCs) on the nrLFA after a pre-determined test time serves as endpoint marker. Upon adding 500 mM CaCl2 solution to citrated bovine, canine, rabbit and equine blood, the average travel distance decreases from 10.9 to 9.4 mm, 8.8 to 5.7 mm, 12.6 to 9 mm, and 15.3 to 11.3 mm, respectively. For heparinized bovine and rabbit blood, the average distance decreases from 14.5 to 11.4 mm and from 9.8 to 7.2 mm, respectively, when adding 300 mg/l protamine sulfate solution. The effect of hematocrit on RBC travel distance in the nrLFA was also investigated. The nrLFA device will potentially improve treatment efficiency, patient safety, quality of life, and satisfaction for both animal patients and their owners.

Reduction of the Adhesive Friction of Elastomers through Laser Texturing of Injection Molds

It is well known that elastomers usually possess poor dry sliding friction properties due to their highly adhesive character. In order to overcome this problematic behavior in industrial applications, interfacial materials such as oils, greases, coatings, or lacks are normally used in order to separate or to functionalize the contact surfaces of elastomers. Alternatively, the high adhesion tendency of elastomers may be explicitly reduced by modifying the elastomer composition itself or by enabling a reduction of its effective contact area through, for example, surface laser texturing. This second approach, i.e., the reduction of the adhesive character of elastomers through laser structuring, will be the main topic of the present study. For this purpose, different micro-sized grooved structures were produced on flat injection molds using an ultra-short pulsed laser. The micro-structured molds were then used to produce injection molded micro-ridged Liquid Silicone Rubber (LSR) sample pads. The investigations consisted firstly of determining the degree of replication of the mold micro-structures onto the surface of the LSR pads and secondly, to ascertain the degree of reduction of the friction force (or coefficient of friction) of these micro-ridged LSR pads in comparison to the benchmark (unstructured LSR pads) when tested under dry conditions against Aluminum alloy (Al-6082) or PA6.6-GF30 plates. For this second part of the investigation, the normal force (or contact pressure) dependency of the coefficient of friction was determined through stepwise load increasing friction tests. The results of these investigations have shown that the production of micro-ridged surfaces on LSR pads through laser structuring of the injection molds could be successfully achieved and that it enables a significant reduction of the friction force for low normal forces (or contact pressures), where the component of adhesion friction is playing an important and determining role in the overall friction behavior of the LSR elastomer.

Functional electrospun nanofibers for multimodal sensitive detection of biogenic amines in food via a simple dipstick assay.

Electrospun nanofibers (ENFs) are promising materials for rapid diagnostic tests like lateral flow assays and dipsticks because they offer an immense surface area while excluding minimal volume, a variety of functional surface groups, and can entrap functional additives within their interior. Here, we show that ENFs on sample pads are superior in comparison to standard polymer membranes for the optical detection of biogenic amines (BAs) in food using a dipstick format. Specifically, cellulose acetate (CA) fibers doped with 2mg/mL of the chromogenic and fluorogenic amine-reactive chameleon dye Py-1 were electrospun into uniform anionic mats. Those extract cationic BAs from real samples and Py-1 transduces BA concentrations into a change of color, reflectance, and fluorescence. Dropping a BA sample onto the nanofiber mat converts the weakly fluorescent pyrylium dye Py-1 into a strongly red emitting pyridinium dye. For the first time, a simple UV lamp excites fluorescence and a digital camera acts as detector. The intensity ratio of the red to the blue channel of the digital image is dependent on the concentration of most relevant BAs indicating food spoilage from 10 to 250μM. This matches the permitted limits for BAs in foods and no false positive signals arise from secondary and tertiary amines. BA detection in seafood samples was also demonstrated successfully. The nanofiber mat dipsticks were up to sixfold more sensitive than those using a polymer membrane with the same dye embedded. Hence, nanofiber-based tests are not only superior to polymer-based dipstick assays, but will also improve the performance of established tests related to food safety, medical diagnostics, and environmental testing. Graphical Absract ᅟ.

FRET on lateral flow test strip to enhance sensitivity for detecting cancer biomarker

Fluorescence resonance energy transfer (FRET) between fluorescein isothiocyanate (FITC) and gold nanoparticles (Au NPs) is introduced in the lateral flow strip to detect cancer biomarker CEA with the color and fluorescence dual-readout. Anti-CEA monoclonal antibody coated Au NPs were on the conjugate pad and FITC labelled antibody (FITC-Ab) for CEA was coated on the test line. All the reagents were general in the lateral flow strip or commercially available and no new materials or technique were involved, which make our proposal a more universal method and easier to operate. With the addition of CEA on the sample pad, anti-CEA monoclonal antibody coated Au NPs-CEA-FITC-Ab complex formed on the test line, leading to a megascopic red line and simultaneous quenched fluorescence of FITC via FRET. The visual limit of detection (LOD) through distinguishing red color change was 10ng/mL and the LOD by differentiating fluorescence intensity was 0.1ng/mL, which was two orders of magnitude lower than that without considering fluorescence in the strip. And the linear range changed from 10–80ng/mL to 5–80ng/mL with the analysis of fluorescence change. Meanwhile, the feasibility of our method applied in real clinical samples was also confirmed.

Point-of-care coagulation monitoring: first clinical experience using a paper-based lateral flow diagnosticdevice.

Vitamin K antagonists such as warfarin are the most widely used class of oral anticoagulants. Due to a narrow therapeutic window, patients on warfarin require regular monitoring. Self-testing using point-of-care (POC) diagnostic devices is available, but cost makes this monitoring method beyond reach for many. The main objective of this research was to assess the clinical utility of a low-cost, paper-based lateral flow POC diagnostic device developed for anticoagulation monitoring without the need for a separate electronic reader. Custom-fabricated lateral flow assay (LFA) test strips comprised of a glass fiber sample pad, a nitrocellulose analytical membrane, a cellulose wicking pad, and a plastic backing card were assembled in a plastic cassette. Healthy volunteers and patients on warfarin therapy were recruited for this prospective study. For each participant, a whole blood sample was collected via fingerstick to determine: (1) international normalized ratio (INR) using the CoaguChek® XS coagulometer, (2) hematocrit by centrifugation, and (3) red blood cell (RBC) travel distance on the experimental LFA device after 240s using digital image analysis. RBC travel distance measured on the LFA device using blood samples obtained from warfarin patients positively correlated with increasing INR value and the LFA device had the capability to statistically distinguish between healthy volunteer INR values and those for patients groups with INR≥2.6. From these data, it is predicted that this low-cost, paper-based LFA device can have clinical utility for identifying anticoagulated patients taking vitamin K antagonists who are outside of the desired therapeutic efficacy window.

Development of immunochromatographic colloidal gold test strip for rapid detection of Haemophilus influenzae in clinical specimens.

A simple and fast test strip for simultaneous detection of Haemophilus influenzae, based on the theory of colloidal gold immunochromatography was developed. In this study, monodispersional colloidal gold and gold-labelled polyclonal antibody (AbP6Line) specific to the linear antigenic determinant OMP6 of H. influenzae were prepared, and identified by UV-visible spectroscopy (200-700nm) and transmission electron microscopy. The test strip was assembled on a plastic backing containing a sample pad, a conjugate pad, an absorbent pad and a nitrocellulose membrane, on which test and control lines were stuck. The strip showed us specific recognization of H. influenzae and did not identify other common respiratory pathogens such as Streptococcus pneumoniae, Mycoplasma pneumoniae, Moraxella catarrhalis and Legionella pneumophila. It was shown that the detection limit of the test strip was as low as 1×106 CFU per ml and the whole process can be completed within 10min. The strips could be stored at 4°C for at least 6months without losing sensitivity or specificity. The test strip was specific, simple and convenient for rapid detection of H. influenzae presenting good stability and reproducibility. The test strip provides a simple, convenient and specific tool for the on-site surveillance and diagnosis of H. influenzae infection in clinical samples.

Analytical Tools to Improve Optimization Procedures for Lateral Flow Assays.

Immunochromatographic or lateral flow assays (LFAs) are inexpensive, easy to use, point-of-care medical diagnostic tests that are found in arenas ranging from a doctor’s office in Manhattan to a rural medical clinic in low resource settings. The simplicity in the LFA itself belies the complex task of optimization required to make the test sensitive, rapid and easy to use. Currently, the manufacturers develop LFAs by empirical optimization of material components (e.g., analytical membranes, conjugate pads and sample pads), biological reagents (e.g., antibodies, blocking reagents and buffers) and the design of delivery geometry. In this paper, we will review conventional optimization and then focus on the latter and outline analytical tools, such as dynamic light scattering and optical biosensors, as well as methods, such as microfluidic flow design and mechanistic models. We are applying these tools to find non-obvious optima of lateral flow assays for improved sensitivity, specificity and manufacturing robustness.

Quantitative and Single-step Enzyme Immunosensing Based on an Electrochemical Detection Coupled with Lateral-flow System.

A single-step electrochemical immunochromatography has been developed: the device was based on two pieces of nitrocellulose membrane, a sample pad with anti-mouse IgG antibody labeled with glucose oxidase (GOx-labeled antibody), a conjugate pad with glucose, and a Pt working electrode. Either antibody or antigen was immobilized on the membrane. The addition of a solution containing mouse IgG, a model target, allows for the dissolution of GOx-labeled antibody in the sample pad to form an immunocomplex. The produced immunocomplex was automatically separated by capturing to the antibody immobilized on the membrane with the sandwich structure or by passing through the membrane modified with an antigen for the competitive reaction. The separated GOx label arrived at the conjugate pad with glucose to undergo the enzyme reaction. Hydrogen peroxide generated by this reaction was detected at the Pt electrode prepared on the second nitrocellulose membrane downstream from the conjugate pad. The results demonstrated that the designed immunochromatography can be applied to quantitative detection with a single-step procedure, because both the GOx-labeled antibody for revealing the immunoreactions and the substrate for the enzyme reaction were prepared in the device. Moreover, the initial concentration of the GOx-labeled antibody permitted control of the detectable concentration for mouse IgG.

Determination of cadmium in human urine by colloidal gold immunochromatographic assay

Objective: To establish the method of colloidal gold immunochrom atographic assay for detecting cadmium ions rapidly. Methods: The anti-cadmium ion monoclonal antibody-gold conjugate was labeled on the binding pad, cadmium ion hapten and goat anti-mouse IgG were coated on nitrocellulose membrane as the detection line (T line) and quality control line (C line) respectively. The sample pad, colloidal gold bonding pad, nitrocellulose membrane and absorption pad were orderly assembled on the PVC board to cut into a test paper strip. The qualitative results of the assay were visualized in color. Results: When detecting the human urine cadmium ions, the results were tested qualitativly within 15 minutes. The detection limit was 30 μg/L. No cross-reactivity with other heavy metal ions. The test paper strip could be stored at 4 ℃ for 3 months. Conclusion: The method has the advantages of low cost, strong specificity, good stability and reliable results, and is suitable for rapid screening of cadmium poisoning of enterprise and occupational health.

Development of an indirect competitive enzyme-linked immunosorbent assay and immunochromatographic assay for hydrocortisone residues in milk

ABSTRACTAn anti-hydrocortisone (HDS) monoclonal antibody, 2G8, based on a HDS succinic anhydride derivative hapten, was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic assay for the detection of HDS in milk samples. The half inhibitory concentration (IC50) of the antibody was 0.095 ng/mL, its limit of detection was 0.013 ng/mL, its linear range of detection was 0.026–0.356 ng/mL, and its cross-reactivity with HDS analogs was <5%. In spiked samples and a recovery test, the recovery rates ranged from 92% to 98.5%, indicating the suitability of this ic-ELISA for the analysis of HDS in milk. The immunochromatographic strip had a cutoff value of 2 ng/mL in milk and could be used for the semiquantitative analysis of HDS. When milk samples were added to the sample pad of the strip, a bright test line indicated <0.2 ng/mL HDS, a weak test line indicated 0.2–2 ng/mL HDS, and no test line indicated ≥2 ng/mL HDS. Analysis of HDS in milk samples showed that results acquired by the immunochromatographic assay agreed well with results acquired by ic-ELISA. Thus, the ic-ELISA and strip assay developed in this study rapidly and sensitively detect HDS residues in milk samples.

Using Rapid Diagnostic Tests as a Source of Viral RNA for Dengue Serotyping by RT-PCR - A Novel Epidemiological Tool.

BackgroundDengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping.Methodology/Principal FindingsWe evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype.Conclusions/SignificanceTherefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions.

Integrated, paper-based potentiometric electronic tongue for the analysis of beer and wine

The following manuscript details the stages of construction of a novel paper-based electronic tongue with an integrated Ag/AgCl reference, which can operate using a minimal amount of sample (40 μL). First, we optimized the fabrication procedure of silver electrodes, testing a set of different methodologies (electroless plating, use of silver nanoparticles and commercial silver paints). Later a novel, integrated electronic tongue system was assembled with the use of readily available materials such as paper, wax, lamination sheets, bleach etc. New system was thoroughly characterized and the ion-selective potentiometric sensors presented performance close to theoretical. An electronic tongue, composed of electrodes sensitive to sodium, calcium, ammonia and a cross-sensitive, anion-selective electrode was used to analyze 34 beer samples (12 types, 19 brands). This system was able to discriminate beers from different brands, and types, indicate presence of stabilizers and antioxidants, dyes or even unmalted cereals and carbohydrates added to the fermentation wort. Samples could be classified by type of fermentation (low, high) and system was able to predict pH and in part also alcohol content of tested beers. In the next step sample volume was minimalized by the use of paper sample pads and measurement in flow conditions. In order to test the impact of this advancement a four electrode system, with cross-sensitive (anion-selective, cation-selective, Ca2+/Mg2+, K+/Na+) electrodes was applied for the analysis of 11 types of wine (4 types of grapes, red/white, 3 countries). Proposed matrix was able to group wines produced from different varieties of grapes (Chardonnay, Americanas, Malbec, Merlot) using only 40 μL of sample. Apart from that, storage stability studies were performed using a multimeter, therefore showing that not only fabrication but also detection can be accomplished by means of off-the-shelf components. This manuscript not only describes new paper-based, potentiometric sensors but also according to our knowledge is the first description of an electrochemical paper-based electronic tongue with integrated reference.