Nanoparticles-Based Naked-Eye Colorimetric Immunoassays for In Vitro Diagnostics

Volume 5 • Issue 1 • 1000e133 J Nanomed Nanotechnol ISSN: 2157-7439 JNMNT, an open access journal Immunoassays are the largest class of assays for the detection of protein biomarkers, which play a prominent role in biomedical diagnostics, biosensors and bioanalytical sciences. There has been a rapidly growing interest in naked-eye immunoassays based on the use of nanoparticles (NP) for in vitro diagnostic (IVD) applications [1]. Naked-eye detection, requiring only the human eyes, is foreseen as the critical requirement for the development of next-generation of low-cost IVD kits. The detection of colorimetric product after enzyme-substrate reaction has always been an integral part of conventional Enzymelinked immunosorbent immunoassay (ELISA). But the naked-eye colorimetric detection can only be done reliably at higher analyte concentrations, which makes it unsuitable for ultrasensitive detection. During the last decade, several prospective NP-based strategies have been developed. Although most of these strategies employ gold nanoparticles (AuNPs) [2], there is a continuously evolving trend towards the use of other prospective nanoparticles (such as Ceria NP, graphene, carbon nanotubes) and nanocomposites. NPs form the critical component of naked-eye colorimetric immunoassays as the aggregation of solutions containing low concentrations of NPs displays a clear colour change.

Immunoassays are the largest class of assays for the detection of protein biomarkers, which play a prominent role in biomedical diagnostics, biosensors and bioanalytical sciences. There has been a rapidly growing interest in naked-eye immunoassays based on the use of nanoparticles (NP) for in vitro diagnostic (IVD) applications [1]. Naked-eye detection, requiring only the human eyes, is foreseen as the critical requirement for the development of next-generation of low-cost IVD kits. The detection of colorimetric product after enzyme-substrate reaction has always been an integral part of conventional Enzymelinked immunosorbent immunoassay (ELISA). But the naked-eye colorimetric detection can only be done reliably at higher analyte concentrations, which makes it unsuitable for ultrasensitive detection. During the last decade, several prospective NP-based strategies have been developed. Although most of these strategies employ gold nanoparticles (AuNPs) [2], there is a continuously evolving trend towards the use of other prospective nanoparticles (such as Ceria NP, graphene, carbon nanotubes) and nanocomposites. NPs form the critical component of naked-eye colorimetric immunoassays as the aggregation of solutions containing low concentrations of NPs displays a clear colour change.
The recent demonstration of plasmonic ELISA for the ultrasensitive detection of disease biomarkers by naked-eye colorimetric detection is a highly prospective development [3]. It is a potential immunoassay format for the developing countries and resource-limited settings as it does not require any sophisticated instruments and is based on the generation of colored solutions of characteristics tonality ( Figure 1A). The developed format is based on the linkage of the bio catalytic cycle of enzyme label to the growth of AuNPs in order to obtain blue or redcolored solutions in the presence or absence of the analyte, respectively. The absence of analyte leads to the reduction of Au ions with hydrogen peroxide (H 2 O 2 ) that results in the formation of non-aggregated AuNPs and the characteristic red color of solution. On the other hand, the presence of analyte leads to the consumption of H 2 O 2 by catalase, which results in the formation of aggregated AuNPs that is responsible for the blue color of solution ( Figure 1B). The developed plasmonic ELISA was demonstrated successfully for the detection of prostate specific antigen (PSA) and HIV-1 capsid antigen p24 in whole serum at ultralow concentration of 1 attogram/mL. It detected p24 in the sera of HIV-infected patients with much lower limit of detection (LOD) in comparison to the highly-expensive 'gold standard' i.e. nucleic acidbased test. The lower LOD of plasmonic ELISA is due to the higher efficiency of catalase to fine tune the concentration of H 2 O 2 . It can be used for the low-cost ultrasensitive detection of p24 to diagnose HIV infection in the developing countries. The developed technology can also be incorporated in lab-on-a-chip and microfluidic devices, which will lead to the development of fully-integrated immuno-analytical systems for point-of-care (POC) IVD.
Copper (Cu)-base click chemistry has also been employed extensively for naked-eye readout of immunoassays [4]. Cu-based click chemistry, where Cu acts as a catalyst, leads to high sensitive and specific Cu monoxide (CuO) NP-based immunoassays. The color change in such immunoassays occurs as a result of aggregation of AuNPs in

Nanoparticles-Based Naked-Eye Colorimetric Immunoassays for In Vitro Diagnostics
Sandeep Kumar Vashist* HSG-IMIT -Institut für Mikro-und Informations Technik, Georges-Koehler-Allee 103, 79110 Freiburg, Germany the presence of Cu II with sodium ascorbate as the reductant. Cu II is released into the solution by HCl and detected by the detection mixture comprising of azide-and alkyne-functionalized AuNPs and sodium ascorbate. The developed naked-eye immunoassay strategy was tested for the detection of ovalbumin (OVA) and HIV-1 gp41 antigen. The procedure involves the binding of antigen to the 96-well MTP, which is followed sequentially by the binding of primary antibody and CuO NP-labeled secondary antibody ( Figure 2). Thereafter, the CuO NPs were dissolved by the addition of HCl, which leads to the formation of Cu II ions that were detected with high sensitivity and specificity by click chemistry. The release of Cu II ions leads to the aggregation of AuNPs functionalized with azide and alkyne groups, which changes the color of solution in about 10 min. The developed immunoassay has the same sensitivity and LOD as the absorbance or fluorescence-based MTP readers-based immunoassays. It enables the cost-effective detection of HIV infection without employing any sophisticated instruments.
A personalized food allergen testing platform 'iTube'has also had been demonstrated [5]. It analyses the colorimetric assays performed in test tubes to automatically detect the allergens in food samples using smart phone (Figure 3). It involves a light-weight (~40 g), compact (~22 mm x 67 mm x 75 mm) and cost-effective opto-mechanical attachment, which comprises of a low-cost plano-convex lens, two diffused light emitting diodes (LEDs) to vertically illuminate the test and control tubes that are loaded from the side, and circular apertures to spatially control the imaging field-of-view (FOV). The image of the colorimetric solution in the iTube is taken by the smart phone's camera and subsequently processed by a smart application in <1s to determine the allergen concentration in food samples. The iTube platform was employed to determine the concentration of allergens in several commercially-available cookies containing varying amounts (1-25 part per million, ppm) of peanut. It precisely quantified food allergens in less than 20 min with a detection limit of ~1 ppm. This promising approach provided a reliable smart phone-based technology for the precise detection of analytes based on colorimetric assays.
The naked-eye detection is ideal for analyte detection in resourcelimited settings. But it has a potential drawback that it is inaccurate for quantifying the exact analyte concentration despite having much lower LOD than conventional ELISA [3]. Therefore, the readout of naked-eye immunoassays has been the bottleneck for developing POC  immunoassays. But this has been solved in the last few years by the arrival of smart systems technologies, such as the use of smart phone camera along with the smart application, which enable highly precise colorimetric detection-based quantification of analyte. However, the NP-based technologies still need to comply with the industrial and healthcare requirements [6]. On the other hand, the last decade has witnessed continuous improvement in immobilization chemistries, immunoassay formats and bioanalytical platforms, which have led to significant advances in the field of immunoassays. These advances have been supplemented by the rapidly evolving technology and business trends towards smart phone-based POC devices for immunoassays [7]. NP-based naked-eye immunoassays will certainly play a pivotal role in the development of cost-effective point-of-care IVD in the near future based on their superior analytical performance, novel characteristic features and plethora of applications. Moreover, the development of recently developed and upcoming supporting technologies will contribute effectively to the commercial success and rapidly growing applications of NPs-based naked-eye immunoassays.