Label-free Protein Detection Based on the Heat-Transfer Method—A Case Study with the Peanut Allergen Ara h 1 and Aptamer-Based Synthetic Receptors

Abstract

Aptamers are an emerging class of molecules that, because of the development of the systematic evolution of ligands by exponential enrichment (SELEX) process, can recognize virtually every target ranging from ions, to proteins, and even whole cells. Although there are many techniques capable of detecting template molecules with aptamer-based systems with high specificity and selectivity, they lack the possibility of integrating them into a compact and portable biosensor setup. Therefore, we will present the heat-transfer method (HTM) as an interesting alternative because this offers detection in a fast and low-cost manner and has the possibility of performing experiments with a fully integrated device. This concept has been demonstrated for a variety of applications including DNA mutation analysis and screening of cancer cells. To the best our knowledge, this is the first report on HTM-based detection of proteins, in this case specifically with aptamer-type receptors. For proof-of-principle purposes, measurements will be performed with the peanut allergen Ara h 1 and results indicate detection limits in the lower nanomolar regime in buffer liquid. As a first proof-of-application, spiked Ara h 1 solutions will be studied in a food matrix of dissolved peanut butter. Reference experiments with the quartz-crystal microbalance will allow for an estimate of the areal density of aptamer molecules on the sensor-chip surface.

Optimization and characterization of a flow cell for heat-transfer-based biosensing

“…To overcome convection it is possible to simply invert the setup and heat the cylindrical fluid column inside the flow cell from above which has been done for the detection of DNA mutations and proteins . However, for the detection of macromolecular targets such as cells or bacteria it is necessary to allow the target particles to sediment to the functional interface located on top of the copper heat provider.…”

“…Changes occurring at the interface will alter its thermal resistance, which can be detected by the setup. HTM has been used for a wide range of applications including DNA mutation analysis , small molecule detection , and the detection of proteins , mammalian cells and bacteria .…”

“…In the original design, a large copper heat sink was coupled to a Perspex flow cell to transfer heat to the functional interface. This way, the flow cell suffered from considerable heat loss to the environment and perturbations in the ambient temperature led to an extensive level of noise on the signal . The new design should provide an improved focus of the heat flow through the functional interface, less heat loss through the cell surroundings, lower power consumption, and an improved temperature control within the heat provider.…”

Thermal annealing of gold coated fiber optic surfaces for improved plasmonic biosensing

“…Although still 2 times higher (i.e. 3 nM), a promising result was reported later while employing the heat-transfer technology [44]. However, the same authors showed that the only so-far obtained LOD value of 1 nM was achieved when employing a more complex electrochemical impedance spectroscopy setup [43].…”

Evaluation of different strategies for magnetic particle functionalization with DNA aptamers

“…Additionally, the same Ara h 1 aptamer was previously implemented in a bioassay on an FO-SPR sensing platform [33], acting as a capture receptor on a gold-coated optical fiber, while detection was performed with a gold nanoparticle-labelled anti-Ara h 1 antibody. Most recently, the same aptamer was used for label-free detection of Ara h 1 in heat-transfer resistance measurements [46]. The reported LODs for those two platforms, 15 mg/ mL and 600 ng/mL, respectively, were approximately 40 and 1.5 times higher than the value reported here.…”