A Disposable Saliva Electrochemical MIP-Based Biosensor for Detection of the Stress Biomarker α-Amylase in Point-of-Care Applications

Tânia S C R Rebelo,Laura I G Sousa,Carlos M Pereira,José A Ribeiro,Ana T S C Brandão,António F Silva,Inês M Miranda

doi:10.3390/electrochem2030028

Abstract

The design and synthesis of artificial receptors based on molecular imprinting (MI) technology for the development of a new MIP-based biosensor for detection of the stress biomarker α-amylase in human saliva in point-of-care (PoC) applications is described in this work. The portable electrochemical devices for monitoring α-amylase consists of cost-effective and disposable gold screen-printed electrodes (AuSPEs). To build the electrochemical device, the template biomolecule was firstly immobilized directly over the working area of the gold chip previously activated with a self-assembled monolayer (SAM) of cysteamine (CA). Then, pyrrole (Py) monomer was selected as building block of a polymeric network prepared by CV electropolymerization. After the electropolymerization process, the enzyme was removed from the polymer film in order to build the specific recognition sites for the target enzyme. The MIP biosensor showed a very wide linear concentration range (between 3.0 × 10−4 to 0.60 mg mL−1 in buffer solution and between 3.0 × 10−4 to 3.0 × 10−2 mg mL−1 in human saliva) and low detection levels were achieved (LOD < 3.0 × 10−4 mg mL−1) using square wave voltammetry (SWV) as the electroanalytical technique.

Highlights

Nowadays, one of the main challenges of our society and health systems is the early detection of several diseases, which directly depends on the correct identification of specific disease biomarkers
Α-amylase, an enzyme of ~60 kDa that belongs to a distinctive group of isoenzymes produced in salivary glands for starch digestion, was identified as a promising sensitive biomarker for stress-related changes in the body that reflect the activity of the sympathetic nervous system [2,3]
In order to access the applicability of the prepared molecularly imprinted polymer (MIP) biosensor in clinical context, recovery studies were performed in treated blank human saliva samples spiked with known amounts of α-amylase

Summary

Introduction

One of the main challenges of our society and health systems is the early detection of several diseases, which directly depends on the correct identification of specific disease biomarkers. After (iv) treating the chip surface with extraction solution, a decrease in the Rct was observed This was consistent with the effective removal of α-am431ylase from the imprinted polymer layer, leaving empty the binding sites in the polymeric structure and providing pathways for redox probe diffusion to the electrode surface. ITPhsuusr,fasicme iwlaarseuxspeedriams erneftesrwenecreespyesrtefomrm, wedhearte tohnelyMnIoPna-nspdecNifIiPc isnutrefraaccetsio, nbsutcafonr oNccIPu,rt.hTeheules,ctsriompiolalyrmexepriezraimtioenntosfwPyerme opnerofmoremr etodoaktptlhaeceMaIfPteransudrNfaIcPe sinucrufabcaetsi,obnuwt iftohr pNuIrPe,PthBeS e(alebcsternocpeoolyfmαe-arimzaytliaosne oasf Pteymmploanteo)m. er took place after surface incubation with pure PBS (absence of α-amylase as template)

Surface Characterization by AFM

Selectivity and Application of the MIP-Based Biosensor

Reagents and Solutions

Apparatus

Synthesis of MIP on the AuSPE Surface

Conclusions