Silver-enhanced colloidal gold dip strip immunoassay integrated with smartphone-based colorimetry for sensitive detection of cardiac marker troponin I.

Cardiac troponin I (cTnI) is a specific cardiac biomarker for diagnosis of acute myocardial infarction (AMI). A sensitive and simple point-of-care test (POCT) is still required for early detection of AMI. To address this need, we developed a dip strip assay based on sandwich immunoassay coupled with a silver enhancement system. Pre-incubation and silver enhancement were introduced to the dip strip to increase sensitivity. Due to the catalytic reaction of the silver enhancement solution, the red color of AuNPs changed to dark brown as silver ions precipitated and enlarged the AuNPs. The obtained results were easily seen by the naked eye. For quantitative analysis, the color intensity of the results was analyzed using a smartphone with RGB color picker application. The effects of operating parameters (volume of AuNP-Ab conjugate, volume of sample, incubation time, and analysis time) were investigated and optimized. Under optimal conditions, the limit of detection (LOD) by the naked eye was 0.5 ng/mL. The LOD with silver enhancement was 50-fold lower than without. For quantitative analysis using the smartphone, linearity of detection was observed through the range of 0.5-50 ng/mL (R2 = 0.9952) and the LOD was 0.12 ng/mL. The developed method was successfully applied to detection of cTnI in serum samples, achieving analytical recoveries and %RSD in the ranges of 96.10-119.17% and 2.91-5.13%, respectively. Additionally, this developed assay was not cross reactive with the potentially interfering serum proteins. These results showed the great potential of this dip strip assay as an alternative POCT for detection of serum cTnI.

Thy-AuNP-AgNP Hybrid Systems for Colorimetric Determination of Copper (II) Ions Using UV-Vis Spectroscopy and Smartphone-Based Detection.

A colorimetric probe based on a hybrid sensing system of gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), and thymine (Thy) was developed for easy and rapid detection of copper (II) ions (Cu2+) in solution. The underlying principle of this probe was the Cu2+-triggered aggregation of the nanoparticle components. Color change of the sensing solution (from red to purple) was clearly observed with naked eyes. The experimental parameters, including pH and concentration of tris buffer, thymine concentration and AgNP dilution ratios, were investigated and optimized. Once optimized, the limits of detection were found to be 1, 0.09 and 0.03 ppm for naked eyes, smartphone application and UV-vis spectrophotometer, respectively. Furthermore, determination of Cu2+ was accomplished within 15 min under ambient conditions. For quantitative analysis, the linearity of detection was observed through ranges of 0.09−0.5 and 0.03−0.5 ppm using smartphone application and UV-vis spectrophotometer, respectively, conforming to the World Health Organization guideline for detection of copper at concentrations < 2 ppm in water. This developed hybrid colorimetric probe exhibited preferential selectivity toward Cu2+, even when assessed in the presence of other metal ions (Al3+, Ca2+, Pb2+, Mn2+, Mg2+, Zn2+, Fe3+, Ni2+, Co2+, Hg2+ and Cd2+). The developed procedure was also successfully applied to quantification of Cu2+ in real water samples. The recovery and relative standard deviation (RSD) values from real water sample analysis were in the ranges of 70.14−103.59 and 3.21−17.63%, respectively. Our findings demonstrated a successful development and implementation of the Thy-AuNP-AgNP hybrid sensing system for rapid, simple and portable Cu2+ detection in water samples using a spectrophotometer or a smartphone-based device.

Heavy metals contamination in soil, surface water, crops, and resident blood in Uthai District, Phra Nakhon Si Ayutthaya, Thailand.

This study evaluates the heavy metal (As, Cd, Cu, Hg, Pb, and Zn) contamination in soil, surface water, and crops in Uthai District, Ayutthaya Province, Thailand, an agricultural area located near an industrial park. Further, the blood levels of these metals in residents living in the study area are investigated. The concentrations of the six metals in soil were below the values permitted under the Soil Quality Standard, Thailand. In contrast, the concentrations of As and Hg in surface water exceeded the permissible limits. For the crops, all heavy metal values in eggplant, kale, and rice were at safe levels. However, in basil, both Hg and Cu levels exceeded the permissible limits, and in coriander, Hg content exceeded the permissible limit. Additionally, the potential health risks of heavy metal exposure through consumption of local crops were assessed using target hazard quotients (THQs) and hazard indices (HIs). The former values of the crops varied. 100.0% for As, 40% for Cd, 60% for Cu, 20% for Pb, and 30% for Zn of the analyzed samples had THQs above 1. This indicated that consumers were probably exposed to some non-carcinogenic health risk (except for Hg which was 0%). Of greater concern, the HI values of each consumed crop were > 1, indicating obvious risk of adverse health effect. Finally, the heavy metal levels in blood from a sample of local residents (n = 16) were assessed along with blood chemistry tests. The levels of all heavy metals were within the normal ranges. Nevertheless, heavy metal contamination in both the environment and food crops raise concerns of health risks to the residents of this area.

Paper-based immunosensor with competitive assay for cortisol detection.

The measurement of cortisol (stress hormone) is important for diagnosis and monitoring of stress-related diseases. A paper-based immunosensor with a competitive assay was developed for quick and easy detection of cortisol levels in serum. The paper-based sensor was fabricated using a simple and cheap wax printing method. Cortisol conjugated-BSA was immobilized on the paper's surface in the detection zone for the competitive immunoassay. Anti-cortisol mAb-conjugated gold nanoparticles, as signal indicator, were used to detect cortisol in the sample. A 2-step procedure included applying the sample and washing for cortisol determination. The results showed that the device had the ability to differentiate cortisol levels into three ranges: < 25 µg/dL, 25-50 µg/dL and > 50 µg/dL by visual detection. The limit of detection determined by an image processing program was 21.5 µg/dL. This assay was successfully developed to detect cortisol in serum, and showed good recovery and precision. Our results correlated well with those obtained using an electrochemiluminescence method. This paper-based immunosensor provided a rapid and simple screening test for serum cortisol detection.

A novel paper-based colorimetry device for the determination of the albumin to creatinine ratio.

A novel paper-based analytical device (PAD) was fabricated and developed for the simple and rapid determination of the albumin to creatinine ratio (ACR) in urine samples. The detection was based on a colorimetric reaction using bromocresol green (BG) in a phosphate buffer (PB) at pH 4 for the determination of albumin (AL) and creatinine (CR). BG changes color from greenish-yellow to bluish-green in the presence of AL and/or CR. Picric acid (PA) in 0.25 M NaOH was used to detect CR, and PA changes color from yellow to orange. Under the optimal conditions, the working range was 10 to 350 mg dL-1 with a detection limit of 7.1 and 5.4 mg dL-1 for AL + CR and CR detection, respectively. The repeatability was evaluated, and the %RSD value was less than 8.23 (n = 10). The ACR was obtained by calculating the AL and CR colorimetric results. Finally, the proposed devices were applied to the determination of AL, CR, and ACR in urine samples. The results obtained by the developed PADs were in good agreement with the standard method and demonstrated the method could reliably measure AL, CR, and ACR. The proposed method provides a low-cost, simple, sensitive, and promising tool for diagnostic identification assay for chronic kidney disease (CKD).

Paper-based amperometric sensor for determination of acetylcholinesterase using screen-printed graphene electrode.

A simple paper-based sensor with electrochemical detection was first developed for rapid, selective, and sensitive determination of acetylcholinesterase (AChE). The screen-printed graphene electrode was used as working electrode providing sensitivity for the sensor. The amperometric detection of AChE is based on the determination of thiocholine (TCh) produced from hydrolysis of acetylthiocholine chloride (ATCh) by AChE. To detect TCh, the ATCh immobilized sheet was stacked onto the detection sheet using double adhesive tape, then samples of AChE were dropped on the back side of an ATCh immobilized sheet with only 1min of incubation time. To avoid interference, glutathione (GSH), the potential of 0.5V vs. Ag/AgCl was applied onto a graphene electrode and the current, which depends on AChE concentration, was measured. Under optimized conditions, the limit of detection (LOD) from the experiment of AChE determination was 0.1U/mL with AChE concentration in range of 0.1-15U/mL. The data correlated well with the data obtained using spectrophotometric method. The developed sensor had been successfully applied to detect AChE in blood samples.

Development of competitive lateral flow immunoassay coupled with silver enhancement for simple and sensitive salivary cortisol detection.

Cortisol is known as a stress biomarker. The measurement of cortisol levels is an early warning indicator for health conditions and diagnosis of stress-related diseases. Herein, a lateral flow immunoassay using a gold nanoparticle label with a silver enhancement system was developed for the simple, sensitive and rapid detection of cortisol. The developed assay was based on a competitive platform of which cortisol-BSA conjugate was immobilized at the test zone to compete with an analyte. The quantitative analysis was performed using gold nanoparticles (AuNPs) as signal labeling. Sequentially, the silver enhancement solution was applied in order to enhance the sensitivity of the assay with the results easily seen by the naked eye. Using this system, the limit of detection (LOD) was found to be 0.5 ng/mL with a 3.6 fold more sensitive detection than without the enhancement system (LOD = 1.8 ng/mL). The salivary cortisol analysis was in the range of 0.5-150 ng/mL (R2 = 0.9984), which is in the clinical acceptable range. For the semi-quantitative analysis, the intensity color of the results was analyzed using an image processing program. The proposed method was successfully applied to detect cortisol in saliva. In addition, the results from our method also complied with the ones of those obtained by using the commercial enzyme-linked immunosorbent assay (ELISA). This developed assay offers great promise for a non-invasive screening test of salivary cortisol.

Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection.

A dramatic increase in pesticide usage in agriculture highlights the need for on-site monitoring for public health and safety. Here, a paper-based sensor combined with a wet system was developed for the simple and rapid screening of organophosphate (OP) and carbamate (CM) pesticides based on the inhibition of acetylcholinesterase (AChE). The paper-based sensor was designed as a foldable device consisting of a cover and detection sheets pre-prepared with indoxyl acetate and AChE, respectively. The paper-based sensor requires only the incubation of a sample on the test zone for 10 minutes, followed by closing of the foldable sheet to initiate the enzymatic reaction. Importantly, the buffer loading hole was additionally designed on the cover sheet to facilitate the interaction of the coated substrate and the immobilized enzyme. This subsequently facilitates the mixing of indoxyl acetate with AChE, resulting in the improved analytical performance of the sensor. The absence or decrease in blue color produced by the AChE hydrolysis of indoxyl acetate can be observed in the presence of OPs and CMs. Under optimized conditions and using image analysis, the limit of detection (LOD) of carbofuran, dichlorvos, carbaryl, paraoxon, and pirimicarb are 0.003, 0.3, 0.5, 0.6, and 0.6 ppm, respectively. The assay could be applied to determine OP and CM residues in spiked food samples. Visual interpretation of the color signal was clearly observed at the concentration of 5 mg/kg. Furthermore, a self-contained sample pre-concentration approach greatly enhanced the detection sensitivity. The paper-based device developed here is low-cost, requires minimal reagents and is easy to handle. As such, it would be practically useful for pesticide screening by non-professional end-users.

Highly selective and sensitive paper-based colorimetric sensor using thiosulfate catalytic etching of silver nanoplates for trace determination of copper ions.

A novel, highly selective and sensitive paper-based colorimetric sensor for trace determination of copper (Cu(2+)) ions was developed. The measurement is based on the catalytic etching of silver nanoplates (AgNPls) by thiosulfate (S2O3(2-)). Upon the addition of Cu(2+) to the ammonium buffer at pH 11, the absorption peak intensity of AuNPls/S2O3(2-) at 522 nm decreased and the pinkish violet AuNPls became clear in color as visible to the naked eye. This assay provides highly sensitive and selective detection of Cu(2+) over other metal ions (K(+), Cr(3+), Cd(2+), Zn(2+), As(3+), Mn(2+), Co(2+), Pb(2+), Al(3+), Ni(2+), Fe(3+), Mg(2+), Hg(2+) and Bi(3+)). A paper-based colorimetric sensor was then developed for the simple and rapid determination of Cu(2+) using the catalytic etching of AgNPls. Under optimized conditions, the modified AgNPls coated at the test zone of the devices immediately changes in color in the presence of Cu(2+). The limit of detection (LOD) was found to be 1.0 ng mL(-1) by visual detection. For semi-quantitative measurement with image processing, the method detected Cu(2+) in the range of 0.5-200 ng mL(-1)(R(2)=0.9974) with an LOD of 0.3 ng mL(-1). The proposed method was successfully applied to detect Cu(2+) in the wide range of real samples including water, food, and blood. The results were in good agreement according to a paired t-test with results from inductively coupled plasma-optical emission spectrometry (ICP-OES).

Development of automated paper-based devices for sequential multistep sandwich enzyme-linked immunosorbent assays using inkjet printing.

To the best of our knowledge, this is the first report on paper-based devices for automating the sequential multistep procedures of a sandwich-type enzyme-linked immunosorbent assay (ELISA) that require only a single-step application of the sample solution. The device was based on a piece of nitrocellulose (NC) membrane with specially designed channels, where all the reagents are applied at different locations in order to control the fluid travel to the detection region. The inkjet printing method, a simple and low-cost process, was used to create the flow channel and device barrier patterns. The fabricated barrier was found to be an efficient boundary for the liquid along the printed design in the NC membrane, enabling direct control of the reagent flow time. ELISA results were obtained with a single-step sample application. The developed devices (so-called automated paper-based devices) provided a simple procedure for the sandwich ELISA, while reducing assay time and reagent consumption. Colorimetric results were measured using digital camera imaging with software processing. The capability of the method developed herein was successfully used to determine the levels of human chorionic gonadotropin (hCG) by ELISA.

Simple and rapid colorimetric detection of Hg(II) by a paper-based device using silver nanoplates.

This work combines lab-on-paper methodology with nanoparticle science to develop a new tool for the simple and rapid determination of Hg(II). The resulting paper-based device enables measurement of Hg(II) from only 2 µL of sample solution. The color of the nanosilver in the test area immediately changes in the presence of Hg(II), and this change can be monitored by the naked eye. This method exhibits superior selectivity towards Hg(II) compared with the other metal ions tested. Furthermore, the results show a significant increase in the Hg(II) analytical signal when Cu(II) is added to the Ag Nanoplates at the test zone. With digital camera imaging and software processing, which are shown to further improve the quantitative capability of this technique, the linear detection range is 5-75 ppm Hg(II) with a limit of detection of 0.12 ppm. Using a pre-concentration scheme (based on repeated 2 µL applications of the test Hg(II) solution onto the same test zone) reduces the limit of detection to 2 ppb. The technique developed by this study provides a rapid, sensitive and selective detection method for aqueous Hg(II) samples and is especially suitable for remote field and environmental analysis.

Lab-on-paper with dual electrochemical/colorimetric detection for simultaneous determination of gold and iron.

A novel lab-on-paper device combining electrochemical and colorimetric detection for the rapid screening of Au(III) in the presence of a common interference, Fe(III), in industrial waste solutions is presented here. With dilute aqua regia (0.1 M HCl + 0.05 M HNO(3)) as the supporting electrolyte, square wave voltammetry on paper provided a well-defined reduction peak for Au(III) at approximately 287 +/- 12 mV vs Ag/AgCl. Under the optimized working conditions, the calibration curve showed good linearity in the concentration range of 1-200 ppm of Au(III) with a correlation coefficient of 0.997. The limit of detection (LOD) of the proposed method is 1 ppm. Interferences from various cations were also studied. Fe(III) is the only metal that affects the electrochemical determination of Au(III) when present above a 2.5-fold excess concentration of that of the Au(III). To overcome this limitation, a colorimetric method was used to simultaneously detect Fe(III) as a screening tool. The procedure was then successfully applied to determine Au(III) in gold-refining waste solutions. The results are in agreement with those obtained from inductively coupled plasma-atomic emission spectrometry (ICP-AES).

Electrochemical behaviors of sulfhydryl compounds in the presence of elemental mercury.

In the expression of bioaccumulated elemental mercury (Hg 0) toxicity, the first Hg 0 oxidation step is crucial. Therefore, to clarify the mechanism underlying the interactions of sulfhydryl (SH) compounds and Hg 0 in the present study, we analyzed the oxidation of reduced glutathione (GSH) and L-cysteine (Cys) in the presence of Hg 0 in aqueous solution by cyclic voltammetry (CV). Production of Hg2+ in the reaction mixture was found to increase along with a decrease in free SH residues. CV showed that the oxidation of Cys increased after a 4-h incubation in the presence of Hg(0), but the oxidation of Cys after a 24-h incubation was suppressed. Conversely, GSH oxidation increased with incubation time in the absence of Hg(0). In the presence of Hg(0), the oxidation of GSH was suppressed. The different reactivities of Cys and GSH with Hg(0) may arise from differences in their oxidation/reduction potentials and pH. The important SH compound interactions with Hg(0) oxidation were as follows: (i) oxidation of Hg(0) to form either mercurous ion (Hg+) or mercuric ion (Hg2+) which both form stable complexes in aqueous solution as Hg I (RS) or Hg II(RS)2; (ii) catalyzed oxidation of SH compounds in the presence of Hg 0; and (iii) suppression of the oxidation of SH compounds due to the reduced concentration of free SH compounds through the binding of SH compounds with Hg+ or Hg2+ The present results demonstrate the chemical reaction processes by which Hg 0 dissolves in aqueous solution in the presence of SH compounds, and contribute to our understanding of SH compounds in non-enzymatic Hg 0 oxidation in vivo.

Electrochemical behaviors of native and thermally denatured fish DNA in the presence of cytosine derivatives and porphyrin by cyclic voltammetry using boron-doped diamond electrode.

The electrochemical behaviors of native and thermally denatured fish DNA was investigated using boron-doped diamond (BDD) film electrode by cyclic voltammetry. The BDD electrode afforded us to measure weak current less than muA for the DNA solution in 100 microl. The mixture of acetic acid and sodium acetate solution (0.2 M) was used as a supporting electrolyte. Two oxidation peaks were observed at about +1.1 V and +1.3 V at pH 4.6 for thermally denatured fish DNA. This is due to the oxidation of guanine and adenine in the denatured fish DNA, respectively. In contrast, the native fish DNA showed ill-defined peaks at +1.1 V. Furthermore, the electrochemical behaviors of thermally denatured fish DNA were studied in the presence of cytosine, cytidine, cytidine-5-monophosphate, tetrakis(1-methypyridinium-4-yl)porphyrin (H(2)(TMPyP)(4+)) and Ru(II)(TMPyP)(4+). The oxidation peak intensity at +1.1 V gradually decreased with the increase of the concentrations of the above compounds. Based on the above studies, electrochemical behaviors of the thermally denatured fish DNA at BDD electrode is discussed.