Lastly, if it wasn't for the McNair Scholars Program and Ramiro Frausto, I would not have been introduced to the idea of graduate school and would have been very lost in the application process. As such, rapid and appropriate malaria diagnosis is necessary to prevent mortality.4 The development and spread of malaria RDTs in endemics. The test and control lines become visible to the naked eye when the antigen-Ab complex and unbound reporter elements bind to these sections of the assay, respectively.
The resulting colorimetric signals appear due to the aggregation of the gold nanoparticles (AuNPs) at these sites. Cross-reactivity and other challenges in multiplex diagnostic development While singleplex LFAs are common in the diagnostic and healthcare worlds, multiplex LFAs exist largely in academic research settings and rarely make it to the clinical setting.9 This is largely due to the difficulties associated with to meet. SAFE or SAFE diagnostic criteria while maintaining a single manufactured test that could be administered without the need for heavy laboratory equipment and trained personnel. A 2012 study analyzing cross-reactivity in multiplex sandwich assays introduced these interaction possibilities as "responsibility pairs", where the number of these interactions increased proportionally to the square of the number of targets in a multiplex assay as shown by the following:
Due to the small size of a typical LFA and the limited chemistry to modify nitrocellulose (the most commonly used membrane for LFAs), there are limited methods to overcome cross-reactivity challenges.
LFA divided into sections before aqua regia digestion for ICP-OES measurement. (SP
First, the visual indicator at the test line must be proportional to the concentration of the analyte in the sample. Third, all of the visual indicators must initially be entrained by the flow at the conjugate pad and none must be captured non-specifically at locations other than the test and control lines of the lateral flow strip. 150 μL of running buffer from the corresponding manufacturer was added to the sample pad of the assay and allowed to flow.
Digital frame-by-frame analysis was performed in ImageJ to identify the leading edge of the fluid front on the LFAs.66 The distance from the sample path to that of the fluid front was measured in pixels and converted to millimeters using the in-frame ruler as a reference. Each section of the LFA was cut by hand with stainless steel razor blades (Figure 7), resulting in eight sections: sample pad (SP), conjugate pad (CP), the first part of nitrocellulose (NC1), test line (TL), the second part of nitrocellulose (NC2), control line (CL), the third part of nitrocellulose (NC3) and wicking pad (WP). Solutions of aqua regia were prepared using 3 parts HCl to 1 part HNO3 (v/v) and 0.667 ml of the mixture was added to each tube for gold dissolution.
The amount of gold extracted from each section of the LFA was quantified using a Perkin Elmer Optima 7000 DV ICP-OES (Perkin Elmer, Waltham, MA, USA). The minimum amount of gold required for visual detection was calculated using (Equation 2), where 𝑟 is the radius of the gold nanoparticle, 𝜌 is the actual density of the colloidal gold solution, 𝑉 is the injection volume of the sample, and 𝑇𝐿 !" is the gold concentration found on the test line at the lowest parasite concentration (Figure 13). The LOD for both the LFR and ICP-OES was calculated using 3σ/κ, where σ is the standard deviation of the blank and κ is the slope is of the calibration curve.
The mean and standard deviation for each section of the LFA for each concentration was calculated. A coefficient of variation (CV) was calculated using (σ/m)*100, where σ is the standard deviation and m is the mean of the data set. The total gold content was calculated by adding up the amount of gold found on each of the constituent sections.
Time (sec)Fluid Front Distance from Sample Pad (mm)
In this area, the buffer flows from the sample pad to the conjugate pad, where it resuspends the dried gold conjugate. From there, the gold conjugate is pressed onto a nitrocellulose membrane, where it is ultimately visualized in the LFA assay window. The front fluids on the B-brand LFA were the first to appear from the sight window, followed by the C-brands and finally the A-brands.
They analyzed the time to reach the location of the test line (indicated by the dashed line in Figure 8) and found that the gold conjugate from the Brand B tests reached the test line in about 9 seconds, which was faster than Brand A (14 seconds) and Brand C (17 seconds). The intensity of the test and control lines was then analyzed by LFR (Figure 9). The area under the intensity line scans from the LFR for the test line signal increases as the parasite concentration in the sample increases (Figure 10).
Over the range of concentrations evaluated (0 p/μL – 800 p/μL), the signal area of the test line is approximately linearly proportional to the analyte concentration.
Position from base of test (mm)
An LOD for this method was calculated to be 130 p/µL (indicated as a horizontal dashed line in Figure 10), which is similar to other literature reports.29,68-70 The data show a directly proportional relationship between parasite concentration and test line intensity. As expected, parasite concentration and test line areal intensity were also shown to be directly proportional for these brands. The only observable difference was the clearance of blood on the nitrocellulose membrane in brand B leading to a reduced test line area compared to brands A and C (Figure A2).
The amount of gold present in the conjugated pathway of an unused LFA was first analyzed for each brand. Since this is the only place where conjugate is deposited, this value represents the total amount of gold found on each LFA. The conjugate path contains the most gold for all brands (Figure A4), as expected.
Brand B contained 72% more gold than brand A and 44% more gold than brand C, highlighting the variation in proprietary formulations of the LFAs. The relatively small amount found just outside the conjugate pad is likely a result of the physical overlap between the conjugate and sample pads, rather than an indication that the tests were stored incorrectly. To evaluate intra- and inter-brand manufacturing variability, 15 additional conjugate pads were cut from unused tests and the gold content was analyzed by ICP-OES.
These data were combined with the conjugate pads from the previously unused tests to obtain a total of 18 samples for all three brands (Figure 11). There is some variation in gold content among the 18 samples with a CV of 13.5%, illustrating the variation between assays. Comparison of CV values between brands shows that brand C has higher test-to-test variability compared to brands A and B.
Brand A Brand B Brand C0.0
Conjugate Pad Samples
PCT; and C) NGAL
Therefore, buffers were compared using test line intensities for PCT assays. I would like to thank Gavin Ward for his contribution to the optimization of the CRP ELISA. When trying to overcome the cross-reactivity challenges that specifically involve paper-based diagnostic assays, there are some limitations given the nature of the assay material.
The membranes were then placed in an oven at 125 °C for 5 minutes so that the wax melted through the thickness of the membrane. Triplex dipstick test images for CRP, PCT, and HRP2 were taken with an iPhone 11 Pro in a well-lit room. The images were then inverted and the line tool was used to draw a line the width of the test (a known distance of 12 millimeters) to change the scale from pixels to millimeters.
To examine whether the conjugation method for forming AuNP-Ab conjugates had an impact on the performance of dipstick assays, a covalent conjugation method was compared with passive adsorption, specifically with the selected PCT detection antibody (HM563) as this antibody which appears to be accumulating in the CRP test line. Multiplex assays performed in the presence of PCT and HRP2 showed good discrimination, with little or no false positives for the detection of other analytes. The test was first performed with a range of HRP2 concentrations and only aHRP2 AuNP-Ab conjugate to determine the success of the assay.
An ELISA confirmed that the false positive is the result of PCT capture antibodies forming a sandwich complex with the CRP antigen and the CRP detection antibody. Several methods were used to improve the penetration of the wax both through the conjugate pad and. Each assay was then placed in a reservoir with 300 μL of 50 mM borate buffer which initiated the movement of conjugates to the assay.
Possible modifications of paper-based multiplex immunoassays are limited due to the inherent nature and size of the assay. Additional conjugate pad materials can also be screened to assess what enables the most thorough melting of the wax.
LFA Section
Point of Care Diagnostics in Resource-Limited Settings: A review of the present and future of PoC in its most needed setting. Point-of-Care serodiagnostic testing for early stage Lyme disease using a multiplexed paper-based immunoassay and machine learning. Simultaneous multiplexed detection of protein and metal ions using a colorimetric microfluidic paper-based analytical device.
A paper-based device with an adjustable time controller for the rapid determination of tumor biomarkers. Multicolored Au/Ag nanoparticles for multiplexed lateral flow assay based on spatial separation and color co-localization. Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays.
Development of a Paper-Based Lateral Flow Immunoassay for Simultaneous Detection of Lipopolysaccharides from Salmonella Serovars. Detection platforms for point-of-care testing based on colorimetric, luminescent and magnetic assays: an overview. Quantification of the number of gold nanoparticles in the test zone of lateral flow immunoassay strips.
Uncovering the Hook Effect: A Comprehensive Study of the Effects of High Antigen Concentration on Sandwich Lateral Flow Immunoassays. Rapid Multiplex Detection of 10 Foodborne Pathogens with a 10-Channel Lateral Flow Assay Based on Phosphorus Conversion Technology. A novel polymer-based nitrocellulose platform for the implementation of a multiplexed paper-based microfluidic coupled immunosorbent assay.
