Glowstick-inspired smartphone-readable reporters for sensitive, multiplexed lateral flow immunoassays

Abstract

The COVID-19 pandemic has increased demand for point-of-care (POC) screening tests such as lateral flow assays (LFAs) and highlighted the need for sensitive and cost-effective POC diagnostic platforms. Here, we demonstrate an LFA platform using standard fluorescent nanoparticle reporters in which optical excitation is replaced by chemical excitation using the peroxyoxalate-based chemistry of inexpensive, shelf-stable glowsticks. The one-step chemi-excitation of fluorescent particles produces visible light readable by an unmodified smartphone, enhancing sensitivity while preserving simplicity and cost-effectiveness. Our Glow LFA detected the common model analyte human chorionic gonadotropin with a limit of detection (LoD) of 39 pg/mL—over ten times more sensitive than standard gold nanoparticles using the same antibodies. We also demonstrate its application to the detection of SARS-CoV-2 nucleoprotein at 100 pg/mL in nasal swab extract. Multiple fluorescent dyes can be chemi-excited by a single reagent, allowing for color multiplexing on a single LFA strip with a smartphone camera. The detection of three analytes on a single LFA test line was demonstrated using red, green, and blue fluorescent reporter particles, making glow LFA a promising platform for multiplexed detection.

Accelerated Development of a COVID-19 Lateral Flow Test in an Academic Setting: Lessons Learned

“…In the presence of target analyte, fluorescent reporter particles bearing analyte-specific detector antibodies are sandwich-bridged to the membrane-bound capture antibodies and accumulate in lines to indicate a positive test. Application of glow stick chemiexcitation reagents makes the particles glow brightly with no excitation light or optical filters (adapted from ref ). This article is licensed under a Creative Commons Attribution 4.0 International License .…”

Advancements and future directions in cardiac biomarker detection using lateral flow assays

“…Beyond the development of single-target LFA, several groups have explored the possibility to apply sensors capable of detecting multiple analytes in a single step. 14 Among those relevant to cardiac disease, it is important to mention an excellent review by Mani et al 47 covering a range of multiplexed cardiac biosensors from the years 2017–2022. In addition, Liu et al 105 found that quantum dots embedded into nanoparticles can be more stable than single Quantum Dots for application in lateral flow systems.…”

“…11–13 As it will be further described in detail, the underlying principle behind these biosensors relies on capillary action and, typically, immunochemical reactions. 14 On this basis, diverse types of samples, including sweat, 15 saliva, 16 and blood, 17 can migrate without the assistance of any external forces through various zones of the LFA strip (sample pad, conjugate pad, test line, control line, etc. ), rendering in a rapid color change in the test line if the target analyte is present.…”

Supra-Quantum Dot Assemblies to Maximize Color-Based Multiplexed Fluorescence Detection with a Smartphone Camera

“…To date, examples of multiplexing have been limited to only two or three targets. 10,13,15,16 Here, we evaluated the level of color multiplexing that is possible with PL imaging on a smartphone camera. Suprananoparticle assemblies of QDs (supra-QDs) that combined red-, green-, and blue-emitting QDs at different ratios within a single colloidal entity were prepared and characterized.…”

“…For PL measurements with smartphone-based devices, the higher brightness of quantum dots (QDs) provides superior analytical performance versus fluorescent dyes by better compensating for the technical limitations of smartphone cameras that were not designed and optimized for this purpose. , Moreover, their narrow, symmetric, and tunable PL emission spectracombined with the ability to induce PL from multiple colors of QDs using a single excitation wavelengthare ideal for multiplexed detection via the red-green-blue (RGB) color channels of smartphone cameras. , The recent development of supra-nanoparticle and super-nanoparticle assemblies of QDs now offers this multiplexing advantage with even greater brightness, permitting immunofluorescent single-cell detection and flow cytometry on a smartphone. − These recent developments lead to the question: What level of color multiplexing is possible with PL imaging on a smartphone? To date, examples of multiplexing have been limited to only two or three targets. ,,, …”