James P. Chambers1,*, Bernard P. Arulanandam1, Leann L. Matta2, Alex Weis3, and James J. Valdes4




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Curr. Issues Mol. Biol. 10: 1–12.


Online journal at www.cimb.org



Biosensor Recognition Elements

James P. Chambers1,*, Bernard P. Arulanandam1, Leann L. Matta2, Alex Weis3, and James J. Valdes4



Department of Biology, The University of Texas at San

Antonio, San Antonio, TX 78249, 2Fischell Department

of Bioengineering, Chemical and Nuclear Engineering,

University of Maryland, MD 20742, 3OncoVista, Inc., San

Antonio, TX 78245, 4Office of the Scientific Advisor for

Biotechnology, Aberdeen Proving Ground, MD 21010,


Introduction

Molecular recognition is central to biosensing. Since the

first biosensor was developed by Updike and Hicks (1967)

many biosensors have been studied and developed.

As shown in Fig. 1, a biosensor can be defined as

a “compact analytical device or unit incorporating a

biological or biologically derived sensitive ‘recognition’

element integrated or associated with a physio-chemical

transducer” (Turner, 2000). Initially, biosensor recognition

elements were isolated from living systems. However,

many biosensor recognition elements now available are

not naturally occurring but have been synthesized in

the laboratory. The sensing of targets, i.e. analytes of

interest, is already being influenced by the emergence of

engineered binding proteins (Feltus and Daunert, 2002).

Employing the techniques of modern biotechnology, it is

now possible to construct DNA polynucleotides at will,

thus opening new paths for generation of biosensor

recognition elements arising from paths not taken by

nature. The following review is restricted to a selective

overview of molecular recognition elements, including

receptors, enzymes, antibodies, nucleic acids, molecular

imprints and lectins currently impacting biosensor

development (Fig. 1). With the advent of nanostructures

and new interface materials, these recognition elements

will be major players in future biosensor development.

“Transduction” of the biorecognition event constitutes

a separate and obviously important area of biosensor

development beyond the scope of the present review.


Receptors

For purposes of biosensing, receptors are alluring because

of their generic “receiving” as well as “sending” functions.

In addition to their being mediators of physiological

processes, receptors are natural targets for a variety of

toxins as well as drugs. Receptors are transmembrane

(plasma and intracellular membranes) and soluble

proteins that bind to specific molecules called ligands,

the binding event initiating a specific cellular response.

Ligand-induced receptor conformational changes give

rise to subsequent events such as channel opening,

adenyl/guanyl cyclase mediated second messenger

generation, and reaction cascades involving a multitude

of other proteins, including G proteins, tyrosine kinases,

phosphatases, phosphorylases, transcription factors, and

antigen processing cell receptor responses all constituting

“transduction” in response to the initial ligand binding


Early on, Valdes and coworkers recognized the

usefulness of receptor preparations as biosensor sensing

elements for a variety of ligands of interests (Valdes et

al., 1988; 1990). Although receptor preparations are

attractive biosensor recognition elements due to high

ligand specificity and affinity, their low yield and relative

instability, labor intensive isolation and lengthy purification

protocols of membrane associated proteins, as well as

transduction difficulties have significantly impeded pursuit

of receptor mediated sensing. However, with the advent

of recombinant techniques and a multitude of expression

systems, generation of large amounts of receptor protein

is now possible, alleviating many of earlier logistical

issues. Direct monitoring of receptor–ligand interaction

was challenging due to absence of signal amplification

associated with other sensor biorecognition elements, for

example, enzyme recognition elements. However, the more

recent development of very sensitive, direct monitoring of

the binding event is now possible using surface plasmon

resonance (Subrahmanyam et al. 2002).



Fig. 1 Configuration of a biosensor showing biorecognition, interface, and transduction elements.

*For correspondence: James.Chambers@utsa.edu






1

USA

event.




2 Chambers et al.


Enzyme based recognition

Catalytic enzyme based sensor recognition elements

are very attractive for biosensor applications due to a

variety of measurable reaction products arising from

the catalytic process, which include protons, electrons,

light, and heat. The enzyme urease has been widely

used as a sensor biorecognition element due to a need

for urea determination/monitoring for both medical and

environmental applications (Barhoumi et al., 2006).

The very apparent inherent, regulatory nature of

allosteric enzymes affords great potential for use as

biosensor recognition elements. The regulatory subunit

functions as the recognition element affecting, either in

positive or negative fashion via conformational changes,

the catalytic site serving as the transducing element

(O’Connell and Guilbault, 2001). Although very attractive,

allosteric proteins are multimeric in nature, presenting

stability as well as expression difficulties. However,

Villaverde and coworkers have successfully engineered

catalytic proteins exhibiting sensing elements in the

form of specific ligand binding sites which, in allosteric

fashion, affect the respective catalytic events in response

to different effectors (O’Connell and Guilbault, 2001). An

engineered enzyme biorecognition element has been

used for detection of HIV antibody in serum entailing two

overlapping epitopes (P1 and P2 from the gp41 envelope

glycoprotein) inserted adjacent to active

site resulting in a hybrid enzyme (Ferrer-Miralles et

al., 2001). A number of engineered allosteric catalytic

biorecognition elements are now available and include

 alkaline phosphatase, and

neural protease (O’Connell and Guilbault, 2001).

In similar fashion, green fluorescent protein (GFP)

is now used in many “allosteric-like” sensing element

applications. Because the fluorophore is an intrinsic part

of the GFP polypeptide chain, no covalent modification

of the protein is required. Numerous sensor applications

involving use of GFP have been described (Doi and

Yanagawa, 1999). Doi and Yanagawa have made hybrid

fusion GFP containing specific molecular recognition

sites by inserting protein domains containing the desired

molecular recognition, binding site into a GFP surface

loop. Random mutation of the ligand specific fusion

protein insert gives rise to a combinatorial library from

which binding of specific analytes can be selected via

changes in fluorescence arising from the binding event

(Doi and Yanagawa, 1999).

Of all enzyme recognition element based biosensors,

the glucose biosensor is the most widely studied and

acclaimed sensor success story. The importance and

success of the glucose biosensor stems from the

clinical relevance of diabetes, with 17 million people

in the US alone afflicted with this life-long and as yet

incurable disorder. Thus, the medical need for glucose

monitoring has resulted in intensive efforts to develop

glucose sensors and, of particular note, development

of implantable electrochemical glucose sensors (Heller,

1999); bloodless glucose measurement (Roe and Smoller,

1998); microfabricated electrophoresis chips (Wang et al.,

2000); needle microsensing (Wang, and Zhang, 2001);

and engineered protein mediated sensing (Yamazaki et

al., 2000).


Glucose monitoring is in reality no trivial task.

Most monitoring of glucose is not a measure of blood

glucose directly but one reflective of glucose levels in

the interstitial fluid of subcutaneous tissue. The ratio of

blood glucose (BG) to interstitial glucose (IG) is close to

unity as long as the glucose concentration is not changing

rapidly; otherwise one is dealing with a complicated

interplay of glucose physiology and insulin kinetics. Not

surprisingly, sensing of glucose can be most difficult in

the “hypoglycemic” range where the greatest accuracy is

demanded.

Although a variety of glucose sensors are available,

the glucose biosensor has changed little in principle

over the years. As shown in Fig. 2, glucose encounters

an immobilized enzyme and transduction is achieved

amperometrically via an electrode. Currently, most

glucose biosensors utilize glucose oxidase as their

recognition element that catalyzes the oxidation of

glucose to gluconolactone:


glucose + O2→ gluconolactone + H2O2

If oxidation is accomplished using glucose

dehydrogenase (NAD+ prosthetic group), NADH is

produced rather than H2O2. In the above reaction scheme,

the dominant detection approach is electrochemical in

nature where the product (hydrogen peroxide or NADH)

is electrochemically detected by an electrochemical

mediator, e.g. organic dyes such as Prussian Blue and

inorganic redox couples which serve as electron sinks.

An alternative transduction scheme involves the use of

quinoprotein glucose dehydrogenase, which requires

neither oxygen nor NAD+ but orthoquinone cofactors to

oxidize a wide variety of alcohols and amines to their

corresponding aldehydes and ketones. The quinoprotein

glucose dehydrogenase recognition element uses

pyrroloquinoline quinone (PQQ) as a cofactor and is

described below:


glucose + PQQ(ox)→ gluconolactone + PQQ(red)


Fig. 2 Diagram of the glucose sensor showing the electrode configuration,

the polymer barrier deposited onto the working electrode, and the surface

where the enzyme (glucose oxidase) is immobilized.






Biosensor Recognition Elements


A novel and exciting improvement over that shown in

Fig. 2 is that of a molecular “wired” enzyme recognition

element based glucose biosensor developed by Heller

(1990). This sensor is comprised of enzymes (glucose

and bilirubin oxidases) electrically wired with redox

polymers I and II, respectively (Fig. ). Interestingly,

because O2 is a natural cosubstrate of glucose oxidase,

it interferes with glucose assays. Thus, as the O2 partial

pressure is increased, the anodic glucose electrooxidation

current, (i.e. signal) decreases. As shown in Fig. (center

complex), the glassy carbon electrode which consists of

cross-linked glucose oxidase (GOx) and redox polymer I

is well shielded, and O2 is electroreduced. As configured,

almost total electroreductive stripping of O2 from the

solution near the glucose electrooxidizing anode is

achieved so that residual reducible O2 no longer defines

the detection limit. Mano and Heller have successfully

detected glucose at femtomolar concentrations (Mano

and Heller, 2005). Importantly, this molecular “wired”

oxidase format can be used for detection of a variety of

important analytes of clinical relevance such as lactate,

L-α-glycerolphosphate and glutamate.

Advances in the field of micro/nanoelectromechanical

systems (MEMS/NEMS) now offer unique opportunities

for extremely accurate, real time measurement of blood

glucose. MEMS/NEMS have demonstrated unique

advantages such as their small size and integration

into a variety of devices. Among the MEMS platforms,

microcantilevers have been proven to be an outstanding

platform for chemical and biological sensors, with detection

limits as low as femtomolar (Tang et al., 2004). Modified

microcantilevers can recognize target molecules through

specified biological binding which results in deflection of

the cantilever. Yan and coworkers have devised a glucose

oxidase functionalized microcantilever for detection of

glucose (Yan et al., 2005).

Carbon nanotubes are hollow graphitic structures

and are promising for immobilization purposes because

of their significant mechanical strength, high surface

area, excellent electrical conductivity and chemical

stability. The lengths of the nanotubes can range from

several hundred nanometers to several micrometers and

the diameter ranges from 0.2 to 2 nanometers for single

walled structures, and 2 to 100 nanometers for coaxial

multiwalled structures. The subtle electronic properties of

carbon nanotubes are not only attractive with regard to the


Fig.  Schematic diagram of glucose detection by electrooxidation on

a stationary glassy disk with electrically “wired” glucose and bilirubin

oxidases.


development of “wired” enzyme biorecognition elements

but also sensitive determination of physiologically

important peptides such as insulin (Zhang et al., 2005).

Current wired enzyme and peptide sensors are the

forerunners of nanowired arrays that can result in

exquisite sensitivity because they are so small that when

an analyte binds at the surface of the recognition element,

the entire nanowire is affected (Hitt, 2004).

In contrast to catalytic schema, binding of substrate

such as glucose without catalysis is ideal because

substrate-depletion effects, in this case reduction of

substrate concentration, are problematic due to “mass

law” considerations. If the binding event is made the

sole basis for detection, then little analyte is consumed,

avoiding depletion effects arising from catalytic conversion

to product. Importantly, such configurations lead to

significantly smaller sensing elements which in turn can

be arrayed at significantly higher density than that for

substrate to product based sensing. Non-catalytic based

sensing of glucose had its origins in the work of Schultz

and coworkers based upon fluorescence resonance

energy transfer (FRET) between a donor and acceptor

following binding of glucose to glucose binding proteins

(Schultz, and Sims, 1979) but found very limited use due

to reversibility and aggregation problems. More recently,

thermostable glucose binding proteins from Aspergillus

niger (glucose oxidase), Thermoplasma acidophilum

(glucose dehydrogenase) and Bacillus stearothermophilus

(glucokinase) have been engineered to report via

fluorescence and/or fluorescence energy transfer the

binding of glucose in a non-consuming manner where

glucose is bound but not chemically converted to product

(Scognamiglio et al. 2004). Bambot and coworkers have

suggested that implanted, reversible, non-consuming

glucose sensing could potentially be achieved through the

skin using red laser diodes or light emitting diode devices

as the light source (Bambot et al., 1995). Importantly,

the use of inactive apoenzymes for reversible sensing

greatly simplifies the sensing platform as well as expands

the range of metabolically important proteins that can be

used as sensor biorecognition elements.

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