Posts Tagged ‘advanced functional materials’

Stephen J. Williams, Ph.D. Writer, Curator

Rational Design of Allosteric Inhibitors and Activators Using the Population-Shift Model: In Vitro Validation and Application to an Artificial Biosensor.(1)

The population-shift mechanism allows for the re-engineering of biosensors utilizing the concept of allosterism to allow for a structure-based switching on/off capacity into biosensors, “smart-biomaterials, and other artificial biotechnologies.  A fundamental problem in the design of valuable biosensors has been limited number of biomolecules that produce enough signal (for example emission of light, etc.) upon binding to its target.  However this issue has been resolved with the development of biosensors in which target binding is transduced into a quantifiable optical or electrochemical signal after coupling with conformational changes in the receptor (for review see (2)).

There are a few advantages to this biosensor design:

  • Works well in complex samples, such as blood, serum; Low background noise from nonspecific adsorption from interfering biomolecules
  • Supports real-time monitoring- allosteric biosensors do not rely on additional reagents and are rapidly reversible
  • Binding of the receptors is dependent on an unfavorable conformational change, so there is possibility to fine tune this conformational switch.

Concept of allosterism

Allosterism is generally defined as a change in the activity and conformation of an enzyme/protein resulting from the binding of a compound at a site on the enzyme other than the active binding site.  Allosterism plays a critical role in the control and integration of molecular events in biological systems.  Frequently, allosterism is seen with multisubunit proteins/enzymes, where subunit interaction is necessary for allosteric effects, and is distal to the binding site.  Examples of allosteric systems include hemoglobin, phosphofructose kinase and many

NAD+ -dependent dehydrogenases.  For example, the binding of O2 to hemoglobin is enhanced the binding of addition O2, the Bohr effect (the affinity of hemoglobin to O2 depends on H+), and the metabolic product diphosphoglycerate regulates O2 binding.

Types of DNA Biosensors

DNA-based biosensors rely on the hybridization of complementary DNA.  Many optical biosensors based on the phenomenon of surface plasmon resonance (SPR) utilize a property of and other materials; specifically that a thin layer of gold on a high refractive index glass surface can absorb laser light, producing electron waves (surface plasmons) on the gold surface. This occurs only at a specific angle and wavelength of incident light and is highly dependent on the surface of the gold, such that binding of a target analyte to a receptor on the gold surface produces a measurable signal.

Electrochemical biosensors are normally based on enzymatic catalysis of a reaction that produces or consumes electrons (such enzymes are rightly called redox enzymes). The sensor substrate usually contains three electrodes; a reference electrode, a working electrode and a counter electrode. The target analyte is involved in the reaction that takes place on the active electrode surface, and the reaction may cause either electron transfer across the double layer (producing a current) or can contribute to the double layer potential (producing a voltage). We can either measure the current (rate of flow of electrons is now proportional to the analyte concentration) at a fixed potential or the potential can be measured at zero current (this gives a logarithmic response). The label-free and direct electrical detection of small peptides and proteins is possible by their intrinsic charges using bio-functional ion-sensitive field-effect transistors.

Piezoelectric sensors utilize crystals which undergo an elastic deformation when an electrical potential is applied to them. An alternating potential produces a standing wave in the crystal at a characteristic frequency. This frequency is highly dependent on the elastic properties of the crystal, such that if a crystal is coated with a biological recognition element the binding of a (large) target analyte to a receptor will produce a change in the resonance frequency, which gives a binding signal. In a mode that uses surface acoustic waves (SAW), the sensitivity is greatly increased.

Type Biological Element Transducer
OpticalFiber Optics

Surface plasmon resonance

Biomolecular interactionAnalysis

Raman spectroscopy

DNA Optical fiberResonant mirror


SERG probe

Electrochemical DNA Carbon paste electrodes
Piezoelectric     FrequencyAcoustics DNA CrystalsCrystals

The most popular of optical DNA biosensors is molecular beacons, DNA probes containing a fluorescent moiety and a quencher of on the same DNA strand. This probe has an internal complementary sequence so as the DNA folds into a secondary structure, most likely a stem-loop or hairpin structure, so the fluor and quencher are held in close proximity, quenching the fluorescent signal.  Target hybridization opens up the stem-loop structure, thereby emitting the fluorescent signal. A typical molecular beacon probe is 25 nucleotides long. A typical molecular beacon structure can be divided in 4 parts:

  • Loop: This is the 18–30 base pair region of the molecular beacon which is complementary to the target sequence.
  • Stem: The beacon stem is formed by the attachment, to both termini of the loop, of two short (5 to 7 nucleotide residues) oligonucleotides that are complementary to each other.
  • 5′ fluorophore: At the 5′ end of the molecular beacon, a fluorescent dye is covalently attached.
  • 3′ quencher (non fluorescent): The quencher dye is covalently attached to the 3′ end of the molecular beacon. When the beacon is in closed loop shape, the quencher resides in proximity to the fluorophore, which results in quenching the fluorescent emission of the latter.

Structure of a molecular beacon. Description and figure from Wikipedia (5).

Common applications of DNA biosensors include cDNA microarray and Affymetrix GeneChip™ technology.

Ricci et al. provide a proof-of –principle paper to demonstrate how allosteric switching can be introduced into biosensors(1). The authors engineered allosteric inhibition into a molecular beacon by the addition of two single-stranded tails that serve as an allosteric site where binding of an inhibitor sequence would bridge the two tails and prevent target binding (holding the probe in the inactivated state).  Using this approach the authors demonstrated over a three-fold increase in the dynamic range of the beacon.

The authors also demonstrated this effect, with an allosterically activated biosensor in which “allosteric activation was engineered into a molecular beacon using one single-stranded tail as an allosteric binding site.  The activator sequence binding to this tail partially invades the stem, destabilizing the nonbinding state and thus improving the target affinity.”  Thus this population-shift mechanism allows for the design of sensors that can be allosterically activated using activators that destabilize the beacon’s nonbinding conformation, increasing the beacon’s dynamic range without compromising target specificity. Finally the authors suggest that population-shift mechanisms can be engineered into many different types of “switching” biosensors including aptamer-based and protein-based sensors (3,4).

1.            Ricci, F., Vallee-Belisle, A., Porchetta, A., and Plaxco, K. W. (2012) Journal of the American Chemical Society 134, 15177-15180

2.            Vallee-Belisle, A., and Plaxco, K. W. (2010) Current opinion in structural biology 20, 518-526

3.            White, R. J., Rowe, A. A., and Plaxco, K. W. (2010) The Analyst 135, 589-594

4.            Kohn, J. E., and Plaxco, K. W. (2005) Proceedings of the National Academy of Sciences of the United States of America 102,   10841-10845

5.            http://en.wikipedia.org/wiki/Molecular_beacon

Other research papers on Biosensors were published on this Scientific Web site as follows:

Measuring glucose without needle pricks: nano-sized biosensors made the test easy

New Definition of MI Unveiled, Fractional Flow Reserve (FFR)CT for Tagging Ischemia

New Drug-Eluting Stent Works Well in STEMI

Sensor detects glucose in saliva, tears for diabetes testing

Synthesizing Synthetic Biology: PLOS Collections

Competition in the Ecosystem of Medical Devices in Cardiac and Vascular Repair: Heart Valves, Stents, Catheterization Tools and Kits for Open Heart and Minimally Invasive Surgery (MIS)

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Reported by: Dr. V.S.Karra, ph.d

Researchers have created a new type of biosensor that can detect minute concentrations of glucose in saliva, tears, and urine, and might be manufactured at low cost because it does not require many processing steps to produce.

“It’s an inherently noninvasive way to estimate glucose content in the body,” says Jonathan Claussen, a former Purdue University doctoral student and now a research scientist at the U.S. Naval Research Laboratory. “Because it can detect glucose in the saliva and tears, it’s a platform that might eventually help to eliminate or reduce the frequency of using pinpricks for diabetes testing. We are proving its functionality.”

Claussen and Purdue doctoral student Anurag Kumar led the project, working with Timothy Fisher, a Purdue professor of mechanical engineering; D. Marshall Porterfield, a professor of agricultural and biological engineering; and other researchers at the university’s Birck Nanotechnology Center.

Findings are detailed in a research paper published in Advanced Functional Materials.

“Most sensors typically measure glucose in blood,” Claussen says. “Many in the literature aren’t able to detect glucose in tears and the saliva. What’s unique is that we can sense in all four different human serums: the saliva, blood, tears, and urine. And that hasn’t been shown before.”

The paper, featured on the journal’s cover, was written by Claussen, Kumar, Fisher, Porterfield, and Purdue researchers David B. Jaroch, M. Haseeb Khawaja, and Allison B. Hibbard.

The sensor has three main parts: layers of nanosheets resembling tiny rose petals made of a material called graphene, which is a single-atom-thick film of carbon; platinum nanoparticles; and the enzyme glucose oxidase.

Each petal contains a few layers of stacked graphene. The edges of the petals have dangling, incomplete chemical bonds, defects where platinum nanoparticles can attach. Electrodes are formed by combining the nanosheet petals and platinum nanoparticles. Then the glucose oxidase attaches to the platinum nanoparticles. The enzyme converts glucose to peroxide, which generates a signal on the electrode.

“Typically, when you want to make a nanostructured biosensor you have to use a lot of processing steps before you reach the final biosensor product,” Kumar says. “That involves lithography, chemical processing, etching, and other steps. The good thing about these petals is that they can be grown on just about any surface, and we don’t need to use any of these steps, so it could be ideal for commercialization.”

In addition to diabetes testing, the technology might be used for sensing a variety of chemical compounds to test for other medical conditions.

“Because we used the enzyme glucose oxidase in this work, it’s geared for diabetes,” Claussen says. “But we could just swap out that enzyme with, for example, glutemate oxidase, to measure the neurotransmitter glutamate to test for Parkinson’s and Alzheimer’s, or ethanol oxidase to monitor alcohol levels for a breathalyzer. It’s very versatile, fast, and portable.”

The technology is able to detect glucose in concentrations as low as 0.3 micromolar, far more sensitive than other electrochemical biosensors based on graphene or graphite, carbon nanotubes, and metallic nanoparticles, Claussen says.

“These are the first findings to report such a low sensing limit and, at the same time, such a wide sensing range,” he says.

The sensor is able to distinguish between glucose and signals from other compounds that often cause interference in sensors: uric acid, ascorbic acid and acetaminophen, which are commonly found in the blood. Unlike glucose, those compounds are said to be electroactive, which means they generate an electrical signal without the presence of an enzyme.

Glucose by itself doesn’t generate a signal but must first react with the enzyme glucose oxidase. Glucose oxidase is used in commercial diabetes test strips for conventional diabetes meters that measure glucose with a finger pinprick.



Purdue University




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