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A gold nanoparticles (AuNPs) enhancement strategy for lateral flow immunoassays (LFIAs) was previously reported to improve assay sensitivity. The multi-steps of detection resulted in a tedious procedure. In this report, to reduce this limitation, a sequential flow LFIA was introduced to perform the AuNPs enhancement in only single step operation using C-reactive protein (CRP) as a model analyte. The delay and non-delay channels created on nitrocellulose membrane (NCM) using wax-printing method were used to deliver immunoassay and AuNPs enhancement reagents to the detection zone sequentially. For the enhancing step, AuNPs were enlarged by the catalytic reaction of KAuCl4 and NH2OH⋅HCl. Under optimal conditions, a calibration curve was found in a range of 0.1−5 μg⋅mL−1 with a good linear correlation (R2 = 0.9953). The limits of detection (LOD) were found to be 0.1 and 0.001 μg⋅mL-1 for naked eye observation and calculation (3SD/slope), respectively. Furthermore, the device shows no cross-reactivity with common interferences for CRP. The platform was successfully applied to detect CRP in certified human serum samples within 15 min, demonstrating the simplicity and speed of the AuNPs enhancement strategy platform. These promising results suggest that the new device may be used as an alternative tool for CRP detection and other biomarkers for POCT analysis.

A paper-based device, based on the fluorescence switching of water-soluble thioglycolic acid (TGA)-capped CdTe QDs, was developed for the simple, rapid and sensitive detection of organophosphorus (OP) and carbamate (CM) insecticides. A new design platform for the developed device was created, comprising a foldable sheet with a detection zone and a buffer solution loading channel, to simplify the multi-step reaction of a bi-enzyme assay with a signal enhancement system. This device was very useful for fast screening and on-site detection, without requiring many reagents. The bi-enzyme includes acetylcholinesterase (AChE), choline oxidase (ChOx) and TGA-capped CdTe QDs, which were coated on the paper-based device at the detection zone in a separated sheet, starting with an acetylcholine (ACh) substrate coating. After incubation of the sample at the detection zone, the device was closed to initiate enzyme catalyzed hydrolysis of the pre-spotted ACh. Subsequently, the buffer solution was applied to the device, allowing for the pre-spotted chemicals to mix well and importantly, result in signal enhancement. The hydrolysis of ACh was catalyzed by AChE followed by ChOx to yield H2O2, which can quench the fluorescence of the TGA-capped CdTe QDs. In the presence of OP and CM insecticides, the AChE activity was inhibited, resulting in decreased quenching of the TGA-capped CdTe QDs. The change in the fluorescence intensity of the TGA-capped CdTe QDs can be observed using the naked eye under UV-black light. Under optimized conditions, the limit of detection for pirimicarb, dichlorvos and carbaryl were found to be 0.05, 0.01 and 0.01 ppm, respectively, using visual measurements. This developed device was successfully applied to the detection of OP and CM insecticide residues in agricultural products. The data correlated well with the data obtained using gas chromatography-tandem mass spectrometry (GC-MS/MS). This developed device shows good selectivity and sensitivity for the rapid screening of insecticides and could be useful for on-site applications.

A l -cysteine-modified silver nanoplate ( l -cys-AgNPl)-based colorimetric assay was developed for simple and rapid screening for copper (II) ions (Cu2+) in aqueous solution. The assay is based on the Cu2+-induced aggregation of l -cys-AgNPls. In the presence of Cu2+, l -cys-AgNPls were aggregated immediately and the color of solution was distinctly changed from violet to blue within 5 min. This method enabled the quantitative measurement of Cu2+ with sample volumes as low as 5 µL. Under optimized conditions, the limit of detection (LOD) was 1 ppm and 0.34 ppm by naked eye and smartphone with RGB application, respectively. For quantitative analysis, the calibration curve was constructed by plotting the differences in absorbance ratio at wavelengths of 620 nm and 570 nm (A620/A570) of sample and blank as a function of Cu2+ concentration. This assay detected Cu2+ in the range of 0.25 to 6 ppm (R2 = 0.9951) and the LOD was 0.22 ppm. In addition, l -cys-AgNPls exhibited good selectivity for Cu2+. Additionally, the developed method was successfully applied for rapid screening of Cu2+ in real wastewater samples with % recovery in the range of 73.08 to 114.62 and 93.48 to 114.04 by UV-visible spectrometry and smartphone with RGB application, respectively. The results from the developed method were compared with those from atomic absorption spectrometry and no significant difference was found between the two methods. The developed colorimetric method is promising for on-site detection of Cu2+ with high accuracy, good selectivity, and minimal reagent consumption.

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., EXCLI Journal; 17:Doc1198; ISSN 1611-2156

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).

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.