Aptasensor for Quantification of Leptin Through PCR Amplification of Short DNA-Aptamers

Abstract

Protein quantification is traditionally performed through enzyme-linked immunosorbent assay (ELISA), which involves long preparation times. To overcome this, new approaches use aptamers as an alternative to antibodies. In this paper, we present a new approach to quantify proteins with short DNA aptamers through polymerase chain reaction (PCR) resulting in shorter protocol times with comparatively improved limit of detection. The proposed method includes a novel way to quantify both the target protein and corresponding short DNA-aptamers simultaneously, which also allows to fully characterise the performance of aptasensors. Human leptin is used as a target protein to validate this technique, because it is considered an important biomarker for obesity-related studies. In our experiments we achieved a lowest limit of detection of 100 pg/mL within less than two hours, a limit affected by the dissociation constant 1 of the leptin aptamer, which could be improved by selecting a more specific aptamer.Because of the simple and inexpensive approach, this technique can be employed for Lab-On-Chip implementations and for rapid 'on-site' quantification of proteins.

A Point-of-Care Device for Fully Automated, Fast and Sensitive Protein Quantification via qPCR

“…As shown in Figure 2 , the system is composed three main modules: a sample preparation module (A), a thermocycler/DNA amplification module (B) for qPCR and a sensing and data processing module (C). The system automates the aptamer-based quantification method depicted in Figure 3 and in our previous publication [ 18 ], which is briefly described in the next section.…”

“…The aptamer-based method for ultrasensitive protein quantification is described in detail in [ 18 ] and briefly presented here. The aptamer and the adaptor are single-stranded DNA, each one being complementary to the other so that they are loosely bound by complementary base pair bonding to form a complex.…”

“…The biochemical assay was developed and validated with human salivary leptin. Details of the validation methods can be found in [ 18 ]. PeakFluo and the CNN were trained and validated on leptin standards quantified through the biochemical assay.…”

“…In this paper, we expand on the work presented in [ 17 ] around a point-of-care device for sensitive protein detection, but compared to our previous works, here we describe the integration of our novel aptasensor (presented in [ 18 ]) with a fully automated POC device. At the basis of the POC device, there is the amplification of the chemical signal through qPCR, which allows accurate detection of minimal protein concentrations.…”

Biological applications of chiral inorganic nanomaterials

“…This kind of chiral sensor exhibited good correlation, with a linear detection range of 0.05–10 ppb, and the LOD was 0.02 ppb, which can satisfy the requirements of the Food and Drug Administration (FDA) for monitoring the BPA content in water. To further enhance the sensitivity of abovementioned chiral sensor, aptamers 108 were anchored on the surface of Au NPs and achieve improvements of the LOD (Figure 6C,D). 62 The linear detection range was 0.02–5 ng ml −1 , and the LOD was 0.008 ng ml −1 , which indicated the chiral aptamer sensor possessed higher specificity and sensitivity than the immuno‐recognition‐driven sensor.…”