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Caroline Cardonnel, PhD | European Applications Supervisor | Molecular Devices
Juliette Lajoux, MSc | Research Engineer, Biology | Poly-Dtech
Susana Brun, PhD | Biology Manager | Poly-Dtech
Mohamadou Sy, PhD | Chemistry Manager | Poly-Dtech

Introduction

Lateral flow assays (LFAs) are widely utilized in diagnostic applications due to their rapid response time, ease of use, and cost-effectiveness. However, conventional LFAs are often constrained by limitations in sensitivity, specificity, and quantitative accuracy, typically providing only qualitative results. To address these challenges, advanced technologies are required to enhance LFA performance and expand their diagnostic capabilities.

This study demonstrates how the integration of Molecular Devices’ ScanLater® Western Blot Detection System with Bright-Dtech™ nanoparticles, a proprietary technology from Poly-Dtech, effectively overcomes the intrinsic limitations of conventional LFAs. Originally developed for high-sensitivity fluorescence detection in western blotting, the ScanLater® system can be repurposed to significantly enhance lateral flow testing, particularly when combined with Bright-Dtech™ technology.

Bright-Dtech™ nanoparticles offer a distinct advantage for LFAs due to their unique lanthanide-based probes, which provide exceptional brightness, photostability, and minimal background interference—critical factors for precise and sensitive fluorescence-based detection. These nanoparticles ensure robust signal intensity, enabling accurate quantification even at low analyte concentrations.

By integrating the ScanLater® system’s quantitative fluorescence measurement capabilities with the enhanced signal stability and brightness of Bright-Dtech™ probes, this approach extends the analytical performance of LFAs beyond their traditional constraints. To illustrate the potential of this combined technology, we present a lateral flow assay specifically developed for the quantitative detection of human lactate dehydrogenase (h-LDH). h-LDH is a key enzyme involved in cellular metabolism, and its quantification serves as an important biomarker of tissue damage. Elevated h-LDH levels indicate cell membrane disruption and are associated with conditions such as hemolysis, myocardial infarction, liver diseases, and cancer. In oncology, LDH is also used as a prognostic marker, with higher levels often reflecting tumor progression and poor outcomes.

This example highlights the increased sensitivity, accuracy, and reliability achieved through this innovative approach, demonstrating its applicability to high-precision diagnostic development and testing in various research and laboratory settings.

Figure 1. Depiction of the LFIA assay for detecting h-LDH using a non-competitive assay format with the Bright-Dtech™-614 Eu nanoparticles (EuNPs) as signal reporters. When excited at 340 nm, these EuNPs emit a red fluorescent light at 614 nm that is detected in a time-resolved mode.

Advantages of Bright-Dtech nanoparticles + ScanLater technology

• Quantitative lateral flow assay: The integration of Bright- Dtech™ nanoparticles with ScanLater® technology enables precise quantification of fluorescence intensity, allowing for accurate determination of analyte concentrations through the test line-to-control line (TL/CL) ratio.
• High sensitivity: Bright-Dtech™ nanoparticles enhance detection sensitivity due to their extended fluorescence lifetime, enabling the reliable quantification of low analyte concentrations.
• Reduced background noise: The time-resolved fluorescence (TRF) capabilities of ScanLater® technology significantly reduce background interference, resulting in clearer and more accurate signal detection.
• Superior sensitivity compared to conventional lateral flow assays: The combined use of Bright-Dtech™ nanoparticles and ScanLater® technology enhances analytical performance, surpassing the sensitivity of traditional lateral flow assays.
• Multiplexed analysis: The ScanLater® system allows for the simultaneous analysis of multiple test strips, increasing throughput and efficiency in high-volume diagnostic workflows.
• Versatile applications: This technology is adaptable to a wide range of analytes and sample types (plasma, serum, saliva, cells, water, etc.), making it suitable for diverse applications across research, veterinary, or even agri-food settings.

Assay principle

Using Bright-Dtech™ nanoparticles, we developed a dipstick lateral flow assay for the quantitative detection of an analyte, using h-LDH as a model. In this assay, Bright- Dtech™ lanthanide nanoparticles replace conventional detection probes, such as gold nanoparticles or traditional europium chelates, to achieve highly sensitive detection with enhanced fluorescence properties.

The test strip consists of a nitrocellulose membrane with two primary detection lines: the test line (TL) and the 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 generating a fluorescent signal proportional to the analyte concentration. The CL serves as an internal control, ensuring assay validity by capturing excess nanoparticles and producing a fluorescence signal independent of analyte presence (Figure 1).

To accurately quantify the fluorescence signal at both the TL and CL after migration, we repurposed the ScanLater® module, originally designed for high-sensitivity fluorescence detection in Western blot analysis. The system accommodates up to 20 strips simultaneously, enhancing assay throughput and consistency. The SpectraMax® iD5 Multi-Mode Microplate Reader, equipped with the ScanLater system and time-resolved fluorescence (TRF) detection, is particularly well-suited for this application, as its optimized fluorescence reading parameters align with the properties of Bright-Dtech™ nanoparticles.

Note: The iD5 reader has been updated to a newer model, the SpectraMax iD5e Multi-Mode Reader, which has equivalent features and performance, plus optional SpectraMax aer Gas Mixer and advanced shaking features.

Fluorescent 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). This configuration significantly reduces background noise from autofluorescence and other short-lived emissions, particularly those originating from the nitrocellulose membrane, leading to a clearer and more reliable quantitative readout.

The fluorescence intensity at the TL and CL is quantified using ImageJ (National Institutes of Health, Bethesda, MD), leveraging the “gel analysis” tool. This tool enables the selection of each strip and transforms band intensities into peak profiles, with the area under the curve corresponding to fluorescence intensity. To standardize results and facilitate comparisons across different assays, we calculate the test line-to-control line ratio (TL/CL), which serves as a normalized measure of analyte concentration.

Figure 2. Strips for the h-LDH LFA placed on the rack of the ScanLater™ system of the TRF SpectraMax® iD5 reader.

Sensitivity and dynamic range

Calibration curves for detecting h-LDH were generated by dispensing 5 μL of conjugated nanoparticles and 75 μL of serial dilution of h-LDH antigen (ranging from 0 to 40 ng/ mL in migration buffer, n = 4) into the wells of a 96-well plate. The test strips were then immersed and allowed to migrate for 20 minutes before being air-dried.

The strips were aligned in the rack (Figure 2) of the ScanLater® system and scanned.

The images were captured using the ScanLater® system and processed with ImageJ software to extract the data (Figure 3). The resulting data were modeled using a 5-parameter logistic (5PL) model in SoftMax Pro™ software (Molecular Devices), with an R² of 1. The system achieved a limit of detection (LoD) of 38 picograms/mL of h-LDH, which demonstrated sensitivity comparable to that of commercial ELISA tests.

Thanks to the optimization of this test, a preclinical study was conducted by spiking h-LDH into serum. The results obtained, with a recovery rate (R%) ranging from 88% to 121%, demonstrate that the test is robust and enables accurate quantification in a complex matrix such as serum.

Figure 3. (A) Images of the EuNPs-based LFIA strips after detecting serial dilutions of h-LDH in working buffer (B) Calibration curve plot representing the normalized signal in the TL achieved when detecting serial dilutions of h-LDH standard in working buffer with EuNPs-based LFIA (n = 4). Inset showing the calibration curve plot between 0.001 and 10 ng mL-1 of h-LDH.

Figure 4. Recovery results when analyzing h-LDH spiked serum samples with EuNPs-based LFA

Conclusion

In summary, the integration of Bright-Dtech™ nanoparticles and ScanLater® technology into lateral flow assays represents a significant advancement in quantitative testing. The enhanced sensitivity of Bright-Dtech™ nanoparticles, combined with the superior background reduction capabilities of ScanLater® technology, enables the detection of low analyte concentrations with high accuracy. Our dipstick assay for human lactate dehydrogenase (h-LDH) exemplifies this potential, demonstrating a limit of detection comparable to that of traditional ELISA tests while offering the advantages of a rapid and user-friendly format.

Further evidence of the effectiveness of combining Bright-Dtech™ nanoparticles with ScanLater® technology to enhance the sensitivity of lateral flow assays can be found in the following study: J. Lajoux & al.: Breaking the picomolar barrier in lateral flow assays using Bright-Dtech™ 614 – Europium nanoparticles for enhanced sensitivity, Microchemical Journal, Volume 209, 2025, 112864. https://doi.org/10.1016/j.microc.2025.112864.

Acknowledgments

We would like to thank all our academic partners and institutes, including the Instituto Interuniversitario de Investigación en Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, through Dr. Amadeo Sena-Torralba, Dr. Ángel Maquieira, and Dr. Sergi Morais; the Institut Pluridisciplinaire Hubert Curien (IPHC, UMR 7178, CNRS/Université de Strasbourg) through Dr. Loïc Charbonnière; and the Grupo de Investigación y Desarrollo en Ciencias, Tecnología e Innovación (BioGRID), Sociedad de Doctores e Investigadores de Colombia (SoPhIC), through Dr. Yulieth D. Banguera-Ordoñez.

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