Advanced trap lateral flow immunoassay sensor for the detection of cortisol in human bodily fluids

Abstract

Paper-based biosensors based on lateral flow immunoassay (LFI) are promising candidates for POC diagnosis because of their ease of use and rapid target detection. However, the low sensitivity of LFI limits its application, and signal amplification has been used in numerous studies to increase its sensitivity. We developed an advanced trap LFI (α-trapLFI), a simple-to-use sensor, with an additional step for signal amplification. Here, signal amplification is automatically implemented following delayed release of enhancement solution induced by water-soluble polyvinyl alcohol tape. As the polyvinyl alcohol tape is exposed to water, its polymer structure is perturbed (within 5 min), allowing ions to pass through. This new sensor was designed to have a short time delay between the flow of solutions used for the immunoassay and signal amplification. The α-trapLFI was subsequently used to detect cortisol with high sensitivity (9.1 pg∙mL^-1) over a broad detection range (0.01-1000 ng∙mL^-1) in bodily fluids. Furthermore, an excellent correlation was obtained by analyzing 20 human real saliva samples using this sensor and a conventional ELISA (R² = 0.90). The new sensor will be helpful in detecting various small molecules for simple, rapid, and portable POC diagnosis of stress disorders.

Scheme 1

Schematic illustration of the new α-trapLFI sensor. (a) An digital image and schematic illustration of α-trapLFI sensor which consists of sample pad, conjugate pad, signal amplification pad, PVA tape, absorbent pad, and detection and deletion zone on membrane. (b) Signal amplification pad is placed on water-soluble polyvinyl alcohol (PVA) tape. After injecting sample and signal amplification solutions consecutively into the sensor, the detection antibody-gold nanoparticles conjugate passes first (the release of the gold ions is delayed by the PVA tape). After the conjugate passes, the PVA tape dissolves allowing the diffusion of ions, thus increasing the signal of the conjugate. (c) Comparison of conventional competitive LFI and α-trap LFI; although conventional LFI sensors can detect the target at high concentrations, the α-trapLFI sensor can detect the target at both low and high concentrations. This is due to two reasons: i) simultaneous and automatic signal amplification and ii) the ratiometric calculation with deletion and detection zones.

Figure 1

Images of polyvinyl alcohol (PVA) tape observed by (a,b) Scanning electron microscope and (c,d) optical microscopy. (a,c) Prior to loading solution; and (b,d) after loading solution. The surface of the PVA tape exposing to water is pitted, and collapsed structure and sizable holes were observed (dotted circles). The SEM images are 60° tilted views.

Figure 2

Delayed-release effect of the polyvinyl alcohol (PVA) tape compared with other membranes. (a) Time-dependent images obtained from assays. (b) Intensity plot. All images were obtained using a ChemiDoc XPS + imaging system (Bio-Rad), and the band intensities were measured using Image Lab software (version 6.1, https://www.bio-rad.com/en-uk/product/image-lab-software?source_wt=imagelabsoftware_surl&ID=KRE6P5E8Z). The error bars indicate the standard deviation from three independent experiments.

Figure 3

Comparison of signal intensities from a conventional lateral flow immunoassay (LFI) and a polyvinyl alcohol (PVA) tape-applied LFI. (a) Time-dependent images obtained from assays. (b) Intensity plot. All images were obtained using a ChemiDoc XPS + imaging system (Bio-Rad), and the band intensities were measured using Image Lab software (version 6.1, https://www.bio-rad.com/en-uk/product/image-lab-software?source_wt=imagelabsoftware_surl&ID=KRE6P5E8Z). Error bars indicate the standard deviation from three independent experiments.

Figure 4

Sensitivity comparisons of the conventional lateral flow immunoassay (LFI) sensor and α-trapLFI sensor. All images were obtained using a ChemiDoc XPS + imaging system (Bio-Rad), and the band intensities were measured using Image Lab software (version 6.1, https://www.bio-rad.com/en-uk/product/image-lab-software?source_wt=imagelabsoftware_surl&ID=KRE6P5E8Z).

Figure 5

Sensitivity of α-trapLFI sensor for cortisol determination in human bodily fluids. Cortisol standard in (a) buffer, (b) saliva, (c) serum, and (d) urine. All images were obtained using a ChemiDoc XPS + imaging system (Bio-Rad), and the band intensities were measured using Image Lab software (version 6.1, https://www.bio-rad.com/en-uk/product/image-lab-software?source_wt=imagelabsoftware_surl&ID=KRE6P5E8Z). Error bars indicate the standard deviation from three independent experiments.

Figure 6

Cross-reactivity of α-trapLFI sensor in human bodily fluids. a Structure of cortisol and other steroid hormones. b Selectivity results comparing cortisol and other steroid hormones. All images were obtained using a ChemiDoc XPS + imaging system (Bio-Rad), and the band intensities were measured using Image Lab software (version 6.1, https://www.bio-rad.com/en-uk/product/image-lab-software?source_wt=imagelabsoftware_surl&ID=KRE6P5E8Z). Error bars indicate the standard deviation from three independent experiments.

Figure 7

Human salivary cortisol level measured using conventional ELISA and α-trapLFI sensor. All images were obtained using a ChemiDoc XPS + imaging system (Bio-Rad), and the band intensities were measured using Image Lab software (version 6.1, https://www.bio-rad.com/en-uk/product/image-lab-software?source_wt=imagelabsoftware_surl&ID=KRE6P5E8Z).