Lateral-flow immunoassay (LFA), also known as rapid kits, is one of the promising technologies that have seen great commercial success and made a marked impact on health clinics. LFA utilizes an inexpensive paper-based device to perform rapid and robust screening of biomarkers in complex biological fluids such as saliva and urine.
LFA requires minimal efforts for specimen preparation and assay and has been explored for clinical diagnosis, environmental monitoring, and personal use. LFA chips for human immunodeficiency virus (HIV) and hepatitis C virus screening, for example, have been available for several years, and rapid kits for pregnancy testing can be readily purchased over the counter.
LFAs typically employ a nitrocellulose membrane, where a region is functionalized with capturing antibodies. The membrane is dipped in a liquid specimen (e.g., saliva) for assay, and the target analytes, if present, bind to antibody-coated metallic nanoparticles, such as gold nanoparticles (AuNPs), in the conjugation pad. This complex migrates through the membrane and is stopped when a capturing antibody in the membrane recognizes the complex. This recognition process results in the accumulation of AuNPs in the activated region of the membrane referred to as test line, producing a color change. This change in color can be read out by a naked eye or colorimetric sensor if the quantitative measurement is desired.
Despite its simplicity and cost-effectiveness, the application of the LFA technology has been limited to only a small number of disease biomarkers largely due to its low sensitivity. As the number of biomarkers in the blood is 10-1000 times larger than that in saliva and urine, LFA assay based on blood specimens may offer improved sensitivity. However, people are not comfortable with taking out blood, and this procedure is often accompanied by other issues such as sample contamination and infection.
Fig. 1 Exemplary laser speckle pattern generated by a laser pointer. Credit: Chulmin Joo
In a recent publication, Song et al. developed a novel and simple optical LFA reader technology, termed photothermal laser speckle imaging (PT-LSI), that may allow high-sensitivity detection of biomarkers without taking out blood sample. This method exploits a combination of two well-known physical phenomena, namely laser speckle and photo-thermal response of AuNPs. When one illuminates a sheet of paper with a light beam of a laser pointer, the light would be scattered by the paper in a random direction, and the interference of the scattered light produces a random granular pattern called a speckle (See Fig. 1). This speckle pattern is a result of the interference of the scattered light from the paper and is thus extremely sensitive to the local changes in refractive index and the structural arrangement of the paper.
Fig. 2 Schematic of the PT-LSI sensor. Probe beam illuminates an LFA membrane. The light is scattered through the membrane and the interference of the scattered light produces a speckle pattern on the image sensor. The sensing region is then illuminated by the pumping beam, which is absorbed by the AuNPs. The absorbed light energy is converted into heat, and the resultant temperature increase alters the speckle pattern. Credit: Chulmin Joo
The authors noted that a reaction-completed LFA membrane is also optically diffuse and thus would generate a speckled pattern under the illumination of a laser (780-nm). The wavelength of the light for speckle pattern generation is set to be outside the absorption band of the AuNPs. If The LFA membrane is illuminated by additional light, photothermal excitation light, of which wavelength is in the high absorption band of the AuNPs (e.g., 532-nm), the AuNPs in the sensing region absorb the PT light energy and convert it into thermal energy. This temperature increase alters the refractive index in the vicinity of AuNPs and may deform the LFA membrane, modulating the speckle pattern being measured on the image sensor. The measurement of this speckle pattern modulation enables quantification of AuNPs, which is directly proportional to the number of the biomarkers in the specimen.
The authors demonstrated that the PT-LSI sensor could measure AuNPs in the range of 1.14 × 109 to 1.44 × 1012 NPs/mL on nitrocellulose membranes with a detection limit of 3.26 × 109 NPs/mL, which corresponds to a 125-fold enhancement in detection limit compared with conventional colorimetric sensor (See Fig. 3(a) and (c)). In order to demonstrate its viability in measuring disease-related biomarkers, the PT-LSI sensor was further employed to perform cryptococcal antigen (CrAg) assay. The HIV-related cryptococcal meningitis has led to a substantial amount of deaths in developing countries. Recognizing its significance, LFA kits for CrAg measurement have been developed and recently approved by the US FDA. The authors applied PT-LSI sensors to perform CrAg assay and demonstrated the detection with a sensitivity 68 times higher compared to the colorimetric sensor (See Fig. 3(b) and (d)).
Fig. 3 PT-LSI vs. colorimetric sensors for AuNP-spotted membranes. (a-b) Photographs of AuNP-spotted membranes and CrAg LFA with various concentrations. (c-d) Dose-response curves of PT-LSI and colorimetric sensors for the AuNP-spotted nitrocellulose membrane and CrAg LFA. Error bars represent standard error. The detection limit of the PT-LSI sensor was 125 times and 68 times smaller than that of the colorimetric sensor for the AuNP and CrAg LFA, respectively. Image published with permission from Elsevier from https://doi.org/10.1016/j.bios.2018.06.024.
One of the attractive features of the PT-LSI sensor is that it can operate with any commercial LFA chips with no modification of its chemistry and fabrication process while achieving greater sensitivity. The PT-LSI sensor also features simplicity, portability, and low-cost in implementation and operation; the sensor can be readily built with inexpensive laser pointers and webcam. Development of a portable PT-LSI sensor that can be linked to mobile devices such as smartphones is presently underway by the authors for field-based sensing of various infectious diseases in developing countries.
These results are described in the article entitled Highly sensitive paper-based immunoassay using photothermal laser speckle imaging, recently published in the journal Biosensors and Bioelectronics. This work was conducted by Chulmin Joo, Seungri Song, Suho Ryu, Soochul Kim, Juncheol Shin, and Hyo-Il Jung from Yonsei University, and Seoyeon Choi from Mirimedix Inc.