Lateral flow assays (LFAs) are commonly employed in diagnostic applications owing to their quick response time, user-friendliness, and affordability. However, standard LFAs are frequently limited by inadequate sensitivity, specificity, and quantitative precision, often delivering only qualitative outcomes.
To overcome these problems, advanced technologies must improve LFA performance and broaden their diagnostic abilities.
This article shows how integrating Molecular Devices’ ScanLater® Western Blot Detection System with Bright-Dtech™ nanoparticles, a proprietary technology from Poly-Dtech, successfully addresses the inherent constraints of standard LFAs.
Initially engineered for high-sensitivity fluorescence detection in Western blotting, the ScanLater® system can be adapted to substantially improve lateral flow assessment, especially when paired with Bright-Dtech™ technology.
Bright-Dtech™ nanoparticles provide a distinct advantage for LFAs through their unique lanthanide-based probes, which deliver excellent brightness, photostability, and low background interference, crucial variables for sensitive and precise fluorescence-based detection. These nanoparticles ensure robust signal intensity, enabling precise quantification even at minimal analyte concentrations.
Advantages
- Combining Bright-Dtech™ nanoparticles with ScanLater® technology delivers outstanding sensitivity over standard LFAs
- Accurate fluorescence intensity quantification enables quantitative LFAs with precise analyte concentration determination
- ScanLater® technology allows multiplexed evaluation of several test strips for enhanced efficiency and throughput in high-volume workflows
By integrating the ScanLater® system’s quantitative fluorescence measurement abilities with the improved brightness and signal stability of Bright-Dtech™ probes, this strategy extends LFA analytical performance beyond its traditional constraints.
To demonstrate the utility of this combined technology, this paper presents an LFA developed specifically for the quantitative detection of prostate-specific antigen (PSA).
This example underscores the improved sensitivity, precision, and dependability achieved with this cutting-edge technique, demonstrating its applicability to high-precision assessment in both clinical development and lab environments.
Benefits of Bright-Dtech Nanoparticles and ScanLater Technology
- Quantitative LFA: By integrating Bright-Dtech™ nanoparticles with ScanLater® technology, the system enables accurate fluorescence intensity quantification. This allows analyte concentrations to be determined accurately using the test-line-to-control-line ratio (TL/CL)
- Elevated Sensitivity: Thanks to their prolonged fluorescence lifetime, Bright-Dtech™ nanoparticles improve detection sensitivity, enabling the dependable quantification of minimal analyte concentrations
- Decreased Background Noise: The time-resolved fluorescence (TRF) abilities of ScanLater® technology substantially decrease background interference, delivering clearer and more precise signal detection
Figure 1. Schematic illustration of the LFA structure for the detection of PSA using a dipstick assay format. Image Credit: Molecular Devices UK Ltd
- Better Sensitivity Compared to Standard LFAs: The combined use of Bright-Dtech™ technology improves analytical performance, exceeding the sensitivity of standard lateral flow assays.
- Multiplexed Evaluation: The ScanLater® system supports the concurrent evaluation of multiple test strips, improving efficiency and throughput in high-volume diagnostic workflows.
- Flexible Applications: This technology can adapt to a wide variety of analytes and sample types (plasma, serum, saliva, cells, water, etc.), making it well-suited for various applications across clinical development, research, veterinary, or even agri-food settings.
Assay Principle
A dipstick LFA for quantitative analyte detection was developed using Bright-Dtech™ nanoparticles, with PSA as a model. In this assay, Bright-Dtech™ lanthanide nanoparticles replace standard detection probes, such as gold nanoparticles or conventional europium chelates, to achieve high-sensitivity detection with improved fluorescence characteristics.
The test strip consists of a nitrocellulose membrane featuring two main detection lines: a test line (TL) and a control line (CL). The TL contains immobilized antibodies specific to the target antigen, which bind the analyte if present in the sample, capturing the nanoparticle-labeled complexes and producing a fluorescent signal proportional to the analyte concentration.
Serving as an internal control, the CL ensures assay validity by capturing excess nanoparticles and generating a fluorescence signal regardless of analyte presence.
For accurate quantification of the fluorescence signal at both the TL and CL following migration, the ScanLater® module, originally developed for high-sensitivity fluorescence detection in Western blot evaluation, was repurposed. The system can handle up to 20 strips concurrently, improving assay consistency and throughput.
The SpectraMax® iD5 Multi-Mode Microplate Reader, equipped with the ScanLater® system and featuring time-resolved fluorescence (TRF) detection, is ideally suited for this application, as its optimized fluorescence-reading parameters align with the luminescence characteristics of Bright-Dtech™ nanoparticles.
Note: The iD5 reader has been upgraded to a newer model, the SpectraMax iD5e Multi-Mode Reader. This newer model possesses equivalent features and performance, as well as optional SpectraMax aer Gas Mixer and advanced shaking functions.
Fluorescence images of the lateral flow strips are acquired in TRF mode with a 0.05 ms delay between excitation (350 nm) and emission (616 nm) measurements. This configuration substantially minimizes background noise from autofluorescence and other short-lived emissions, especially those originating from the nitrocellulose membrane, resulting in a clearer and more dependable quantitative readout.
The fluorescence intensities at the TL and CL are quantified using ImageJ (National Institutes of Health, Bethesda, MD) with the “gel analysis” tool. This tool allows each strip to be selected and converts band intensities into peak profiles, with the region below the curve corresponding to fluorescence intensity.
To standardize findings and support comparisons across assays, the test-line-to-control-line ratio (TL:CL) is calculated, providing a normalized measure of analyte concentration.
Sensitivity and Dynamic Range
Calibration curves for PSA detection were produced by dispensing 5 μL of conjugated nanoparticles and 75 μL of serial dilutions of PSA antigen (ranging from 0 to 300 ng/mL in migration buffer, n = 4) into the wells of a 96-well plate. The test strips were then immersed and left to migrate for 20 minutes before air drying.
Figure 2. Strips for the PSA LFA placed on the ScanLater™ system of the TRF SpectraMax® iD5 reader. Image Credit: Molecular Devices UK Ltd
The strips were aligned in the ScanLater® system rack (Figure 2) and scanned. Additional images were visually assessed for the limit of detection (LoD), which was compared to results acquired with ScanLater® technology (Figure 3).
The images were captured using the ScanLater® system and processed in ImageJ for data extraction. The resulting data was modeled using a five-parameter logistic (5PL) model in SoftMax Pro™ Software (Molecular Devices) (Figure 4).
The system obtained a limit of detection (LoD) of 15 picograms/mL of PSA, which outperformed the visual LoD considerably and demonstrated sensitivity comparable to that of commercial ELISA tests.
This sensitivity, alongside the dependability of the reading mode, enabled patient plasma samples to be successfully quantified.
The results obtained are comparable to those obtained using the reference method (a direct two-site sandwich chemiluminescent immunoassay (Siemens Healthineers)) used by medical analysis labs (Figure 5), further demonstrating the effectiveness of the ScanLater® module for reading Bright-Dtech™ lateral flow tests.
Figure 3. Typical images captured with a smartphone and a 595 nm filter under UV light, and with the SpectraMax iD5 reader, using a twofold serially diluted PSA standard in migration buffer and a negative control (0 ng/mL PSA). White and yellow stars indicate the limits of detection. Image Credit: Molecular Devices UK Ltd
Figure 4. The calibration curve plot of (B) representing the normalized test line by control line (T/C) signal (n=4). Image Credit: Molecular Devices UK Ltd
| Sample |
Reference method
[PSA, ng/mL] |
Bright-Dtech™ LFA
[PSA, ng/mL] |
% Recovery |
| 1 |
<4 |
<LoD |
– |
| 2 |
<4 |
<LoD |
– |
| 3 |
4.9 |
5.5 |
113% |
| 4 |
5.0 |
5.7 |
114% |
| 5 |
5.4 |
6.2 |
114% |
| 6 |
5.4 |
6.1 |
112% |
| 7 |
22.9 |
24.9 |
109% |
| 8 |
23 |
25.5 |
111% |
| 9 |
28.64 |
31.4 |
110% |
| 10 |
38.8 |
37.3 |
96% |
| 11 |
95.5 |
98.2 |
103% |
Figure 5. Comparison of PSA quantification using the reference method and the Bright-Dtech™ LFA. Image Credit: Molecular Devices UK Ltd
Conclusion
To conclude, integrating Bright-Dtech™ nanoparticles and ScanLater® technology into LFAs represents a substantial advancement in diagnostic testing. The improved sensitivity of Bright-Dtech™ nanoparticles, combined with the remarkable background-reduction capabilities of ScanLater® technology, enables high-accuracy detection of low analyte concentrations.
The dipstick assay for PSA exemplifies this potential, exhibiting a limit of detection comparable to that of conventional ELISA tests while delivering the benefits of a fast and easy-to-use format.
Acknowledgments
Produced from materials originally authored by Caroline Cardonnel, PhD, European Applications Supervisor at Molecular Devices; Juliette Lajoux, MSc, Research Engineer, Biology at Poly-Dtech; Susana Brun, PhD, Biology Manager at Poly-Dtech; and Mohamadou Sy, PhD, Chemistry Manager at Poly-Dtech.
About Molecular Devices UK Ltd
Molecular Devices is one of the world’s leading providers of high-performance life science technology. We make advanced scientific discovery possible for academia, pharma, and biotech customers with platforms for high-throughput screening, genomic and cellular analysis, colony selection and microplate detection. From cancer to COVID-19, we've contributed to scientific breakthroughs described in over 230,000 peer-reviewed publications.
Over 160,000 of our innovative solutions are incorporated into laboratories worldwide, enabling scientists to improve productivity and effectiveness – ultimately accelerating research and the development of new therapeutics. Molecular Devices is headquartered in Silicon Valley, Calif., with best-in-class teams around the globe. Over 1,000 associates are guided by our diverse leadership team and female president who prioritize a culture of collaboration, engagement, diversity, and inclusion.
To learn more about how Molecular Devices helps fast-track scientific discovery, visit www.moleculardevices.com.
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