Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

Summary

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

Abstract

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

Introduction

Precise and rapid protein sensing methods are very important in medical diagnostics and proteomics. Classical protein-detecting arrays, such as biochips, are based on the “lock-and-key” recognition principle and require specific receptors such as aptamers, antibodies, or mimetics.

In recent years, differential sensing inspired by the human olfaction and gustation has emerged as an alternative¹. This electronic nose/tongue (eN/eT) approach is based on differential binding of analytes to an array of cross-reactive receptors (CRRs), that do not need to be highly specific or selective for the target molecules thus allow t....

Protocol

1. Preparation of Various Solutions and Protein Samples

1. Prepare 100 ml of phosphate buffer solution (PBS-G) containing 50 mM NaH₂PO₄, 50 mM NaCl, and 10% glycerol at pH 6.8.
2. Prepare 250 ml of HEPES buffer solution containing 10 mM HEPES, 150 mM NaCl, 0.005% Tween 20 at pH 7.4.
3. Prepare stock solution of building block 1 (BB1) lactose and building block 2 (BB2) sulfated lactose (Figure 1) at 0.2 mM in PBS-G.
4. Prepare 1 ml of protein solutions in HEPES: Arachis hypogaea lectin (AHL) at 500 nM, myoglobin at 1 µM, and lysozyme at 500 nM.
5. Prepare 20 ml of 1% SDS in u....

Results

To probe the ability of the electronic tongue for common protein analysis, three proteins were used: AHL, myoglobin and lysozyme. For each protein, a distinct 2D continuous evolution profile, CEP, was generated by the eT, as shown in Figure 6.

In addition, thanks to SPRi, which is able to monitor the real-time adsorption and desorption kinetics, for each protein a time dependent continuous recognition pattern, called 3D continuous evolution landscape (CEL), was generated. In <.......

Discussion

The most critical steps for construction of this eT are dedicated to ensure good reproducibility of the system. For example, cleaning the gold surface of the prism with a standardized procedure before use, adding 10% of glycerol in the eleven pure and mixed solutions of BB1 and BB2 to eliminate solvent evaporation during BBs self-assembling on the gold surface of the prism, depositing multiple replicates for each [BB1]/([BB1]+[BB2]) ratio, etc. As for protein sensing by SPRi, the choice of the working angle.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SPRi apparatus	Horiba Scientific-GenOptics		SPRi apparatus is placed in a temperature regulated incubator at 25 °C.
Incubator	Memmert
Syringe pump	Cavro scientific instruments	Cavro XLP 6000
Micro Elite Degasser	Alltech	AT590507
6-port medium pressure injection valve	Upchurch Scientific		The volume of injection loop used is 500 µl.
Femto plasma cleaner (version 7)	Diener Electronic		On-line degassing system with 2 channel.
Spotter	Siliflow		It is a non-contact piezoelectric spotter.
SPRi-Biochip	Horiba Scientific-GenOptics	36000067	The prism is made of a high refractive index glass prism coated with a thin gold film (45 nm) and specially developed for imaging purposes.
Erythrina cristagalli lectin	Sigma-Aldrich	L5390
Arachis hypogaea lectin	Sigma-Aldrich	L0881
Myoglobin	Sigma-Aldrich	M1882
Lysozyme	Sigma-Aldrich	L6876
CXCL12α			Provided by Dr. Hugues Lortat-Jacob; for preparation details, see supporting information in reference 9
CXCL12γ			Provided by Dr. Hugues Lortat-Jacob; for preparation details, see supporting information in reference 9
Lactose			Provided by Prof. David Bonnaffé; for preparation details, see supporting information in reference 9
Sulfated lactose			Provided by Prof. David Bonnaffé; for preparation details, see supporting information in reference 9
Glycerol	Sigma-Aldrich	G5150
SDS	Sigma-Aldrich	L4509
Tween 20	Sigma-Aldrich	274348
HEPES	Sigma-Aldrich	H3375
Sodium phosphate monobasic	Sigma-Aldrich	S0751
Sodium chloride	Sigma-Aldrich	S3014