The Quartz Crystal Microbalance (QCM) as a Biosensor

Thesis Proposal 
 

Mentors:

Dr. Ruth Baltus

Dr. Linda Luck 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

David Bogdan

March 4, 2005

Introduction

      Biosensors are an increasingly important technology in the detection of compounds ranging from pesticides to biological weapons.  Typically, biosensors consist of a biological macromolecule that is immobilized on the surface of a signal transducer, as shown in Figure 1.  As the macromolecule binds specifically to the ligand being

Figure 1.  Schematic for generic biosensor[1]

detected, the signal transducer can measure a physical change due to the binding event.  The transducer usually detects a change in resistance, pH, heat, light, or mass and then converts that data to an electrical signal to be collected and processed.

Figure 2.   Crystals used in a QCM [2]

 One promising type of detector is the Quartz Crystal Microbalance (QCM).  The QCM is a piezoelectric mass-sensing device.  A QCM device works by sending an electrical signal through a gold-plated quartz crystal (shown in Figure 2), which causes a vibration at some resonant frequency.  The QCM then measures the frequency of oscillation in the crystal.  When used as a biosensor, the QCM can detect changes in frequency of the crystal due to changes in mass on the surface of the crystal.

      While there has been significant research on the use of the QCM as a biosensor, there are many possibilities that have not been explored and many problems that have not been solved.  This study will look at the use of the QCM as a biosensor with the leucine-isoleucine-valine (LIV), leucine-specific (LS) and glucose-galactose receptor (GGR) periplasmic binding proteins of gram-negative E. coli.  The study will also look at the feasibility of using the QCM with a flow-cell, and the effects of temperature on detection using a QCM.

Background

      Gram-negative bacteria have both inner and outer membranes, and it is between these two membranes that periplasmic binding proteins exist.  Over 100 of these proteins have already been discovered and examined by X-ray crystallography [2].  The proteins bind to ligands as either part of active transport systems or as signals for chemotaxis systems [3].  Periplasmic binding proteins are particularly well suited for use in biosensors because they have a high specificity for ligand binding, along with being both soluble and stable [4].  The proteins have a monomeric structure, but have two large lobes that bend on a hinge-like peptide strand [3,4].  Binding occurs by the two lobes closing around the substrate, which is held in by low-energy hydrogen bonds.  By closing around the substrate, the protein makes a conformational change, which is the signal for the active transport or chemotaxis system [3]. 

      This conformational change is the likely explanation for how the quartz crystal microbalance can detect the binding of very small molecules, such as glucose (MW=180.16) or leucine (MW=131.17), when being bound by much larger proteins such as GGR (MW=33,370 [4]) and LIV (MW=36,744 [1]).  It is known that there are a number of interfacial properties, such as mass, the viscosity of the layer, the stiffness of the layer, and the change in conductivity of the layer, that influence the frequency of the crystal vibration, but how this occurs on the molecular level is not yet understood [4].  Determining how the temperature at which the binding event is detected affects the overall shift in frequency of the crystal could help explain whether or not the conformational change of the protein, and therefore the stiffness of the layer, is the key-determining factor in the detection of such small ligands.  If the stiffness of the layer is the key factor, higher temperatures should loosen the proteins, and a smaller frequency shift should be observed.

      For the QCM to measure a binding event, the receptor protein must be attached to the surface of the crystal.  Previous studies have accomplished this by using thiol-containing compounds [4].  The thiocompounds create a self-assembled monolayer on the surface of the crystal, bound by gold-sulfur bonds.  The receptor protein is then covalently bound to the thiocompounds, and an orderly monolayer of the protein is obtained.  Another method that has been used in previous studies here at Clarkson University is genetically Engineering the receptor protein to contain the amino acid cysteine, the