Colorimetric Reagent Research Articles

A loop-mediated isothermal amplification (LAMP) assay for the consensus detection of human pathogenic Campylobacter species

Most rapid identification methods for Campylobacter are designed to detect thermotolerant Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli). A growing number of thermosensitive Campylobacter species are now gaining recognition as emerging human pathogens. Methods are lacking for the rapid screening of these emerging species. Loop-mediated Isothermal Amplification (LAMP) is a nucleic acid amplification method that allows for the rapid and cost-effective detection of bacteria. Degenerate primers against the 16S rRNA sequences for C. jejuni, C. coli, C. lari, C. upsaliensis, C. ureolyticus, C. fetus, C. gracilis, C. rectus, and C. concisus were designed. Isothermal amplification was conducted using ATCC reference strains at 68 °C for 30 min using WarmStart® Colorimetric LAMP reagents. Positive reactions were indicated by a color change from pink to yellow; specificity to Campylobacter was confirmed using a restriction enzyme digest (RsaI). The developed LAMP reaction was specific for the reference strains, which was confirmed against an exclusivity panel that consisted of other enteric pathogens, including E. coli, Salmonella, Shigella, Helicobacter, and Arcobacter. This method was also evaluated for the detection of C. jejuni, C. coli, and C. lari in primary enrichment media from artificially contaminated fresh spinach samples. The LAMP method provides an option to rapidly screen for the presence of pathogenic Campylobacter spp. in field surveillance and trace-back analysis.

A 3D origami paper-based analytical device combined with PVC membrane for colorimetric assay of heavy metal ions: Application to determination of Cu(II) in water samples

Microfluidic paper-based analytical devices (μPADs) as a potentially powerful analytical platform have recently gained significant attention for on-site monitoring of heavy metal ions, which are one of the most significant environmental concern because of non-degradability and high toxicity. The commonly applied μPADs suffers from some defects, such as heterogeneous deposition of reagent, resulting in poor detection limits and low sensitivity. So, in this work, a three-dimensional origami μPAD combined with PVC Membrane was developed, which can manage problems of movement of colored products or leaching out the dye and leading to color heterogeneity in the detection zones. Furthermore, a waste layer was added to μPAD for loading of more amounts of the analyte, which results in improvement of detection limit. As a proof of concept, the μPAD was used for the analysis of Cu2+ ion. For this purpose, pyrocatechol violet and chrome azurol S as colorimetric reagents were doped into PVC membrane and injected in the detection zone. The proposed μPAD was presented good linearity in the ranges of 5.0–1400.0 and 5.0–200.0 mg L−1, and the limits of detections of 1.7 and 1.9 mg L−1 in presence of chrome azurol S and pyrocatechol violet, respectively.

Development of Colorimetric Sensor Array for Instant Determination of Sodium Metabisulfite in Dried Longan

Sodium metabisulfite has been used as a pretreatment chemical to prevent the gradual discoloration and to slow down the spoilage process of dried longan. The excessive use of this sulfiting agent can result in allergic reactions, particularly for sulfite-sensitive people. In this research study, a colorimetric strip was fabricated to determine the concentration of sodium metabisulfite in dried longan. The detection was based on reactions of a series of colorimetric reagents including four different Fuchsine compounds and an Ellman’s regent. The maximum change of color was observed 3 min after the reactions. To perform a quantitative analysis, the image of the strip was captured using a commercial scanner, and it was converted into an RGB color model to be digitally analyzed by chemometrics. Using partial least square (PLS) regression, the calibration range (5.00–250.00 mg/kg) provided R2 and Q2 values of 0.994 and 0.908, respectively. The limits of detection and quantitation were 5.48 and 16.6 mg/kg, which were sensible for the detection of the sulfiting agent in dried longan samples. The developed colorimetric strip was applied to determine the concentration of metabisulfite in dried longan fruit purchased from local markets in Chiang Mai, Thailand. The results agreed well with the findings obtained from the standard reference method. The developed strip could be an alternative solution for rapid, simple, and low-cost detection of the sulfiting agent in dried agricultural products, where test results could be instantly obtained without the requirement of sophisticated analytical instruments.

Cation-exchange strategy for a colorimetric paper sensor: Belt-like ZnSe nanoframes toward visual determination of heavy metal ions

A colorimetric paper sensor based on a cation-exchange strategy has been developed for the visual determination of heavy metal ions. Belt-like ZnSe nanoframes as colorimetric reagents were prepared from ZnSe·0.5N2H4 hybrid nanobelts as precursors via a hydrothermal method. With the decomposition and release of N2H4 ligands, the regular belt-like nanostructures of the precursors evolved into belt-like nanoframes, which consisted of numerous ZnSe nanocrystals. Inspired by the cation-exchange characteristic of chalcogenides, the as-prepared belt-like ZnSe nanoframes were employed to fabricate a colorimetric paper sensor for the detection of heavy metal ions. Owing to the color change arising from a composition change, the colorimetric paper was successfully applied to detect Ag+, Cu2+, and Hg2+ at room temperature. Depending on the color change and its intensity, heavy metal ions of Ag+, Cu2+, and Hg2+ can be individually identified and their concentrations can be determined. Particularly for Cu2+, its visually detected concentration can be down to 1 ppm. More importantly, the fabricated colorimetric paper sensor displays excellent anti-interference properties from various typical cations and anions. Considering its low cost and practicality, expectedly it can be used for practical application.

Chitin Nanofiber Paper toward Optical (Bio)sensing Applications.

Because of numerous inherent and unrivaled features of nanofibers made of chitin, the second most plentiful natural-based polymer (after cellulose), including affordability, abundant nature, biodegradability, biocompatibility, commercial availability, flexibility, transparency, and extraordinary mechanical and physicochemical properties, chitin nanofibers (ChNFs) are being applied as one of the most appealing bionanomaterials in a myriad of fields. Herein, we exploited the beneficial properties offered by the ChNF paper to fabricate transparent, efficient, biocompatible, flexible, and miniaturized optical sensing bioplatforms via embedding/immobilizing various plasmonic nanoparticles (silver and gold nanoparticles), photoluminescent nanoparticles (CdTe quantum dots, carbon dots, and NaYF4:Yb3+@Er3+&SiO2 upconversion nanoparticles) along with colorimetric reagents (curcumin, dithizone, etc.) in the 3D nanonetwork scaffold of the ChNF paper. Several configurations, including 2D multi-wall and 2D cuvette patterns with hydrophobic barriers/walls and hydrophilic test zones/channels, were easily printed using laser printing technology or punched as spot patterns on the dried ChNF paper-based nanocomposites to fabricate the (bio)sensing platforms. A variety of (bio)chemicals as model analytes were used to confirm the efficiency and applicability of the fabricated ChNF paper-based sensing bioplatforms. The developed (bio)sensors were also coupled with smartphone technology to take the advantages of smartphone-based monitoring/sensing devices along with the Internet of Nano Things (IoNT)/the Internet of Medical Things (IoMT) concepts for easy-to-use sensing applications. Building upon the unrivaled and inherent features of ChNF as a very promising bionanomaterial, we foresee that the ChNF paper-based sensing bioplatforms will emerge new opportunities for the development of innovative strategies to fabricate cost-effective, simple, smart, transparent, biodegradable, miniaturized, flexible, portable, and easy-to-use (bio)sensing/monitoring devices.

Post-column detection of cadmium chelators by high-performance liquid chromatography using 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid

Cd(II) is toxic to many species, including humans, because it inactivates a number of enzymes and induces cytopathic effects in the liver, kidney, and skeletal tissues in humans. Metallothionein and glutathione (GSH) play a major role in the protection against Cd(II)-induced toxicity in mammalian cells. In this study, a relatively simple method for detecting trace amounts of Cd(II) chelators was developed by using 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid (TPPS). The TPPS–Cd(II) complex was added to the elutions of high-performance liquid chromatography. The Cd(II) chelators separated by column chromatography were mixed with Cd(II)-bound TPPS (TPPS-Cd(II)). Cd(II) from TPPS-Cd(II) was chelated by the eluted Cd(II) chelators, resulting in the formation of free TPPS. The absorbance of TPPS shifted from 434 nm (TPPS–Cd(II)) to 414 nm (TPPS), and this characteristic shift was used to estimate the quantity and affinity of the Cd(II) chelators. This new method was compared with the bathocuproine disulfonate (BCS) method developed in our previous study. Instead of BCS-Cu(I), TPPS-Cd(II) was used as the colorimetric reagent. The experimental setup of the TPPS-based method is more general, and the preparation of the colorimetric solution is also much simpler than the BCS method. To verify the efficacy of this new method, we determined the actual Cd(II)-chelating ability of GSH in horse blood; the obtained concentration was in good agreement with the previously reported value.

A Colorimetric Artificial Olfactory System for Airborne Improvised Explosive Identification

The detection of ultralow or nonvolatile target analytes remains a significant challenge for artificial olfactory systems even after decades of development, which severely limits their widespread application. To overcome this challenge, an artificial olfactory system based on a colorimetric hydrogel array is constructed for the first time as a universal representative. As an effective extension of conventional artificial olfactory systems that integrates the merits of its predecessors, the proposed system accurately mimics olfactory mucosa and specific odorant binding proteins using hydrogels endowed with specific colorimetric reagents for the detection of hypochlorite, chlorate, perchlorate, urea, and nitrate. Therefore, the proposed system is capable of detecting and discriminating between these five airborne improvised explosive microparticulates with a detection limit as low as 39.4 pg. Additionally, the system demonstrates good reusability over ten cycles, rapid response time of ≈0.2 s, and excellent discrimination properties, despite significant variation. This proof-of-concept study on colorimetric artificial olfactory systems yields a novel strategy for the direct and discriminative detection of nonvolatile airborne microparticulates.

Design and optimization of a single-use optical sensor based on a polymer inclusion membrane for zinc determination in drinks, food supplement and foot health care products.

A single-use optical sensor was designed for Zn(II) determination based on the immobilisation of the colorimetric reagent 2-acetylpyridine benzoylhydrazone (2-APBH) in a polymer inclusion membrane (PIM) adhered on the surface of an inert rectangular strip of polyester (Mylar). Different components for the membrane preparation were tested and those resulting in membrane with good appearance, proper physical and optical properties and ease of preparation were selected. Factorial design 23 with three replicates of the central point was applied for the optimisation of the membrane composition. The optimal composition consisted of 2.5g of poly(vinyl chloride) (PVC), 4mL of tributyl phosphate (TBP) and 0.04g of 2-APBH. The optode showed a linear dynamic range from 0.03 (detection limit) to 1mgL-1 of Zn(II) ions with a response time of 30min in aqueous solution at pH6 and a relative standard deviation of 3.90% for 0.4mgL-1 of Zn(II). The sensor exhibited good selectivity to Zn(II) over other commonly ions. It was successfully applied to the determination of Zn(II) in a water certified reference material, spiked tap water, vitamin-mineral drink, food supplement and foot health care products, as contribution to the concern about this heavy metal due to its significant role in many biological and physiological processes although toxicant at high doses.

The Blepharis persica seed hydroalcoholic extract synergistically enhances the apoptotic effect of doxorubicin in human colon cancer and gastric cancer cells.

The goal of this survey is to evaluate the anti-proliferative effects of the hydroalcholic extract of Blepharis persica seeds and its synergic effect on doxorubicin (DOX) in human colon cancer (HT-29) and gastric cancer cell (AGS) lines. 70% Ethanol was used for extraction of B. persica seed. Aluminum-chloride colorimetric and Folin-Ciocalteu reagent methods were used to measure total flavonoid and total phenolic contents of the extract respectively. Gas chromatography-mass spectrometry (GC-MS) analysis of the B. persica extract was performed on GC-MS equipment after silylation. HT-29, AGS, and human fibroblast (SKM) cell lines were treated by different concentration of the B. persica extract, (DOX) and the combination of extraction and DOX. The cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay while the apoptosis induction was monitored using flowcytometry by annexin-V FITC/PI double-staining. The changes in expression levels of BAX and BCL-2 were determined using Real-Time RT-qPCR. GC-MS analysis of the hydroalcoholic extract from B. persica seeds revealed 24 major components. The MTT assay revealed the cytotoxicity against three cell lines and also it was shown that 125ng/mL of DOX and 0.625mg/mL of B. persica extract had synergistic behavior against HT29 cell line. These results showed B. persica extract induced apoptosis in AGS and HT29 cells and its extract caused dose-dependent increase in up-regulation of BAX level (p < 0.05) and down-regulation of BCL2 (p < 0.05). B. persica showed the synergistic effect in combination with DOX on HT29 cell line. These findings demonstrated a basis for further studies on the characterization and mechanistic evaluation of the bioactive compounds of B. persica extract which had antiproliferative effects on cancer cell lines.

Revisiting the phenanthroline and ferrozine colorimetric methods for quantification of Fe(II) in Fenton reactions

Abstract Colorimetric methods for Fe(II) determination based on complexation of heterocyclic amines, such as phenanthroline (Phen) and ferrozine (FZ), have been widely used for analysis of iron species in solution, which is critical to discuss the mechanism of Fenton and related reaction systems. However, the reliability of both methods in the background of hydrogen peroxide has never been addressed. In this study, we demonstrate that serious Fe(II) overestimation possibly occurs for both methods in Fenton systems, particularly for the Phen method. A systematic variation of incubation conditions reveals that the available Fe(II) concentration depends greatly on the incubation time after mixing the colorimetric reagents with the sample. Also, it is highly sensitive to pH and temperature as well as the initial concentrations of Fe(III), H2O2, and acetate buffer. The experimental results combined with simulations indicate that Fe(II) overestimation mainly results from the reduction of Fe(III)-Phen or Fe(III)-FZ complexes by residual H2O2 during analysis, a reaction that is facilitated by the ligand field effect of the heterocyclic amine. An effective remedy for the instability and inaccuracy of the methods in the presence of H2O2 is the addition of NH4F to the incubation mixture (55 mM for Phen and 35 mM for FZ), which converts residual Fe(III) to a colorless and reduction-insensitive fluoride complex.

Unique coated neusilin pellets with a more distinct and fast visual detection of nerve agents and other cholinesterase inhibitors

Pellets with an immobilized enzyme (acetyl- or butyrylcholinesterase) are the up-to-date type of carriers used for the detection of nerve agents (soman, sarin, tabun, VX, Novichok) and other cholinesterase inhibitors such as organophosphate and carbamate insecticides (parathion, malathion). They are used in the glass detection tubes as a layer containing the enzyme together with the second layer, which contains a colorimetric reagent and substrate. The detection method is based on the visually or spectrophotometrically observable Ellman's reaction, which develops a yellow color in the absence of the cholinesterase inhibitor; otherwise, the detector preserves its original color (preferably white). This reaction occurs very fast and has a high sensitivity to nerve agents but it suffers from an indistinctive color transition from white to yellow. In the presented study, a new approach with the use of the synergic effect of magnesium aluminometasilicate with a high surface area marketed as Neusilin®US2 and a protective semipermeable Eudragit® RL layer was utilized. The prepared pellets have been evaluated for their properties such as the activity of the enzyme, intensity of the developed yellow color, sensitivity to cholinesterase inhibitor physostigmine, which acts as a nerve agent simulant, and physical parameters such as hardness, pycnometric density and sphericity. After the initial evaluation, all samples underwent a stability test under three different storage conditions for 24 months during which they were evaluated at given time points (0, 3, 6, 12 and 24 months). It was found that the prepared samples achieved a much higher intensity of developed yellow color than in the published studies while maintaining similar or better sensitivity, speed of detection and suitable physico-chemical properties.

Development of biodegradable hybrid polymer film for detection of formaldehyde in seafood products

Despite the enormous accomplishments of current sensing methods, portable and sensitive sensing materials remains a challenging issue. Herein, a novel of a biodegradable hybrid polymer film was developed for quantitative analysis of formaldehyde seafood, including Lutjanus erythropterus, Euthynnus affinis, Caranx indicus, and Penaeus monodon at Sabah, Malaysia. In this research, starch and chitosan were introduced as the substrate to entrap Nash colorimetric reagents for the fabrication of biodegradable films for detection of formaldehyde. Under optimal conditions, excellent linearity (R2 = 0.9918) of colorimetric response was obtained in formaldehyde concentration ranges of 100 to 0 ppm, with a limit of detection and quantification calculated to be 5 and 16.8 ppm, respectively. The developed film was successfully applied to the identification and quantification of formaldehyde in four different seafood samples with satisfactory recoveries, and RSD values obtained range between 98.80%–104.65% and 0.12%–1.21%, respectively. The present research demonstrated short response time (within 5 min) that provides reliable methods for application in biosensing, which exhibited the advantage of this well-performing platform for application in the food, environmental, and medical disciplines sensing.

Analytical possibilities of Putrescine and Cadaverine enzymatic colorimetric determination in tuna based on diamine oxidase: A critical study of the use of ABTS

The joint determination of putrescine (Put) and cadaverine (Cad) in the presence of other biogenic amines is studied using their enzymatic reaction with diamine oxidase (DAO). Three alternative methods are studied based on the intrinsic colorimetric properties of DAO or horseradish peroxidase (HRP), and the use of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) colorimetric reagent, respectively. In this last case an in-depth study is carried out in order to explain and solve some drawbacks usually associated with the use of this reagent (especially interferences, interaction with enzymes and instability), and to propose new analytical methodologies which this reagent allows to achieve (transient signal and the use of the violet species). Finally, the method has been applied to Put + Cad determination in a tuna sample without interference of other biogenic amines. The result has been compared with that obtained using a method based on HPLC-MS, which has allowed the new methodology to be validated.

Simple and rapid colorimetric sensing of Ni(II) ions in tap water based on aggregation of citrate-stabilized silver nanoparticles

The presence of nickel in our water sources presents a danger to human health and aquatic organisms alike. Therefore, there is a need to detect and monitor nickel concentration in these sources. Current detection methods use instruments that are costly, resource intensive and require skilled personnel. Alternative methods that are simple and fast are needed. This study reports for the first time the development of a simple colorimetric assay for the detection of Ni2+ in aqueous solution using citrate-stabilized silver nanoparticles. Chemical synthesis method was employed using sodium borohydride and trisodium citrate as reducing and stabilizing agents, respectively. The characterization techniques used for obtaining spectral and morphological properties were ultraviolet-visible spectroscopy and transmission electron microscopy. The resultant silver nanoparticle solution was yellow in color and exhibited an absorbance peak at 392 nm. The full width at half maximum value was 57.8 ± 1.3 nm indicating particle monodispersity. For the morphology, the nanoparticles were spherical in shape with an average size of 10.4 ± 4.5 nm. Furthermore, the colorimetric properties of the silver nanoparticles for Ni2+ detection was investigated. Ni2+ was visually detected through a fast solution color change from yellow to orange. Monitoring of the absorbance showed a decrease in the original 392 nm peak and broadening of the surface plasmon absorption band. These changes are attributed to the aggregation of the nanoparticles due to Ni2+ addition which was confirmed by the transmission electron microscopy imaging. The absorption ratio (A510/A392) was plotted against varying Ni2+ concentration and it exhibited a good linear correlation from 0.7 to 1.6 mM with an R2 of 0.9958. The limits of detection and quantification were 0.75 and 1.52 mM, respectively. Additionally, the assay was tested against other common ions and showed excellent selectivity for Ni2+. Finally, the assay was successfully tested to detect Ni2+ in tap water samples with an average difference of 16% from prepared concentrations. Overall, the study showed the potential of using citrate-stabilized silver nanoparticles as a colorimetric reagent for detecting Ni2+ in aqueous solution.

Determination of Cr(III) and Cr(VI) in water by dual-gel electromembrane extraction and a microfluidic paper-based device

A major goal of green chemistry is to avoid the use of toxic organic solvents. In particular, there are few green methods allowing extraction and quantification of chromium species in environmental samples. Here, we developed home-made, dual-gel electromembrane extraction combined with a microfluidic paper-based analytical device for the simultaneous determination of Cr(III) and Cr(VI) in water, without using any organic solvent. The positively charged Cr(III) and negatively charged Cr(VI) migrated selectively into the cathodic (pH 2.0) and anodic (pH 3.0) aqueous acceptor phases, respectively. After extraction, the anodic acceptor phase containing Cr(VI) was analyzed directly by a microfluidic paper-based analytical device, after the addition of the diphenylcarbazide colorimetric reagent. The cathodic acceptor phase containing Cr(III) was mixed with Ce(IV) to oxidize Cr(III) to Cr(VI), and then, Cr(VI) ions were detected on the microfluidic paper-based analytical device after adding diphenylcarbazide. Under the optimized conditions, limits of detection of 2.0 and 3.0 ng/mL and extraction recoveries of 58.8% and 83.3% were achieved for Cr(VI) and Cr(III), respectively.

Development of ultrafast colorimetric microplate assay method for ubiquinone utilizing the redox cycle of the quinone

A simple, rapid, and sensitive microplate assay method for determination of ubiquinone in cosmetic products has been developed and validated. This method is based on measuring reactive oxygen species (ROS) generated from the redox cycle of quinone using redox colorimetric reagent, 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT). The reaction with ROS converts INT to a formazan dye having a strong absorbance at 510 nm that is increased with increasing concentration of ubiquinone. On the basis of this reaction, we developed a microplate assay method that allows high-throughput measurement of ubiquinone. This method uses microplate reader for the measurement of absorbance and it can determine many samples at one time. Under the optimized conditions, the proposed method was linear in the range of 0.02–4 μM and the limit of detection (blank +3SD) was 14.8 nM. We applied the proposed method to determine ubiquinone contents in cosmetic products, and the measurement values were in good agreement with the indicated value. The proposed colorimetric method should be useful as a high-throughput analysis for quality management of ubiquinone containing products.

Polymethacrylate Sensors for Rapid Digital Colorimetric Analysis of Toxicants in Natural and Anthropogenic Objects

The paper presents a set of measurement (analytical) procedures for a rapid digital colorimetric analysis (DCA) of the following toxicants: tracer agents in drilling liquid; cobalt, chromium, and overall content $\Sigma $ Me of heavy metals (Fe, Cd, Co, Zn, Pb, Ni, Cu, and Mn) in tap water, industrial wastewater and river water; nitrites and $\Sigma $ Me in soils and in vegetables grown in these soils. The analytical procedures were implemented in a computerized electronic device, called digital colorimetric analyzer (DC-analyzer), using specially developed polymethacrylate sensors with the immobilized colorimetric reagents. The experimental validation has shown that the analytical performance (in terms of linear ranges, linearity, sensitivity, trueness, and precision under conditions of repeatability) of the proposed DCA procedures is not worse than the analytical quality of the procedures based on the traditional solid-phase spectrophotometry (SPS) for the same analytes. In terms of response time, the DCA operates (3–4.5) $\times 10^{3}$ times faster than the SPS. The designed DC-analyzer has small weight and dimensions. Together with the developed analytical procedures it can be widely applied for monitoring the sanitary quality of drinking water in a relatively inexpensive and fast way.

Disposable optical sensor for Al(III) ions determination by coupled colorimetric solid-phase extraction-reflectance spectroscopy in leachates from cookware, antacids and hygienic care products

A disposable and miniaturised optical sensor based on colorimetric solid-phase extraction has been designed using poly(styrene-divinylbenzene) membrane disks modified with the colorimetric reagent pyridoxal salicyloylhydrazone to determine the aluminium concentration in aqueous solutions. The extraction of Al(III) ions by the reagent immobilised onto a disk allows the quantification directly on the adsorbent surface by a miniature portable reflectance spectrometer with an optical fibre at 434 nm. The optimisation of the sensing system was carried out by a fractional factorial design 33−1 considering the extraction pH, amount of ligand immobilised onto the disk and time of immobilisation as experimental factors. The linear dynamic range of the sensor response ranged from 0.18 to 2 mg L−1 Al(III) with a detection limit of 0.18 mg L−1 (n = 10), being the precision of 6.3% for 1 mg L−1 Al(III) (n = 10, confidence level of 95%). The proposed method was successfully applied to the analysis of aluminium in leachates from cookware, antacids and hygienic care products, as contribution to the concern about aluminium as a known systemic toxicant at high doses.

Microfluidic Paper-based Analytical Devices (µPADs) For Analysis Lead Using Naked Eye and Colorimetric Detections

This work describes comparation two designs of a microfluidic paper-based analytical devices (µPADs) using a wax printing for fabrication of hydrophobic zones on the chromatographic paper and their application to the detection of lead in waste samples. Two different designs used in this research were distance-based method and analysis image method. Sodium rhodizonate in tartrate buffer solution (pH 2.8) solution are used as the colorimetric reagents for the direct naked eyed detection of lead(II) on µPADs. The detection of lead(II) concentration is conducted by measuring the distanceof a colored reaction product which propagate in the detection zone (design 1) and the change in color intensity on the µPADs is also used for the lead detection using ImageJ software to determine the RGB value (design 2). Related to both methods can be used to determine the concentration of lead(II) with a comparable coefficient of determination, but based on the analysis method, design one is a design that is very simple, easy, fast, inexpensive and only uses the naked eye for detection.

Accessible Telemedicine Diagnostics with ELISA in a 3D Printed Pipette Tip.

We report herein a novel pipet-based "ELISA in a tip" as a new versatile diagnostic tool featuring better sensitivity, shorter incubation time, accessibility, and low sample and reagent volumes compared to traditional ELISA. Capture and analysis of data by a cell phone facilitates electronic delivery of results to health care providers. Pipette tips were designed and 3D printed as adapters to fit most commercial 50-200 μL pipettes. Capture antibodies (Ab1) are immobilized on the inner walls of the pipet tip, which serves as the assay compartment where samples and reagents are moved in and out by pipetting. Signals are generated using colorimetric or chemiluminescent (CL) reagents and can be quantified using a cell phone, CCD camera, or plate reader. We utilized pipet-tip ELISA to detect four cancer biomarker proteins with detection limits similar to or lower than microplate ELISAs at 25% assay cost and time. Recoveries of these proteins from spiked human serum were 85-115% or better, depending slightly on detection mode. Using CCD camera quantification of CL with femto-luminol reagent gave limits of detection (LOD) as low as 0.5 pg/mL. Patient samples (13) were assayed for 3 biomarker proteins with results well correlated to conventional ELISA and an established microfluidic electrochemical immunoassay.