SPECTROstar Nano
Absorbance plate reader with cuvette port
Detectors that are based on biomolecules become increasingly important. This is underlined by the best-known and most profitable biosensor example: the glucose meter. Lesser-known, but highly helpful and popular is the use of biosensors in biological laboratories. Especially life science researchers value the specific, sensitive and quick detection process that led to the development of innumerable applications that are often microplate-based.
What are Biosensors?
A biosensor is defined as an analytical device that combines a biological component with a physicochemical detector. They are used to detect chemical or biochemical compounds. In order to better understand what the practical use of this abstract definition is, the first paragraph will introduce the components of biosensors.
Biological components of biosensors
The biological element of a biosensor directly interacts with the analyte of interest. Popular biosensors use antibodies, enzymes, microbes, or nucleic acids. These recognize the analyzed molecules by specifically binding or converting them.
Physicochemical detector of biosensors
The detector of a biosensor transforms and transduces the signal coming from the interaction of biological elements with analytes into an electrical or optical signal that is used for readout. The so-called biotransducers either measure changes in current, resistance, or charge following a chemical reaction or the change of an optical characteristic such as fluorescence or absorbance. The prime example of a biosensor, today’s glucose meter, is based on an electrochemical transducer. It quantifies blood sugar with the help of an enzyme that converts glucose (e.g. glucose oxidase) and in course of that reaction changes the current which is recorded by an amperometric detector.
Biosensors using optical transducers record changes of light that occur in course of interaction of analyte and biological element. One such change is the refractive index which is measured for surface plasmon resonance applications and is a result of molecules binding to a surface. Another example is a color change that reports on enzyme activity which specifically takes place in the presence of the analyte. Furthermore, biosensors which increase their fluorescence or energy transfer between fluorophores upon analyte exposition are often used in life science laboratories. Mostly, the signal change is detected by a microplate reader. Examples how fluorescent biosensors can be used to measure receptor activation are given below.
Benefits of biosensors
One explanation for the increased use of biosensors is their excellent specificity. This remarkable feature is explained by the biological element exploited for the detection of the molecule of interest. Biological interactions such as antibodies binding to their antigens, the conversion of a substrate by an enzyme or the association of two complementary DNA strands are typically very specific as no organism can afford to initiate unnecessary reaction cascades devouring a lot of energy. For instance, microbial biosensors for harmful arsenic are based on a bacterial arsenic exclusion pathway. In presence of As(III), a transporter will be expressed that exports the toxin out of the microorganism. Instead of the transporter system, the biosensor expresses As(III)-dependently a fluorescent reporter gene. As the elaborate As(III) resistance machinery is only started in the presence of arsenic, its recognition is guaranteed to be very specific in both the organism and in the biosensor.
Another advantage of using biomolecule-based detection is the speed of analysis. The analyte is typically recognized directly and immediately gives a measurable signal. Therefore, biosensors are also commonly embedded in handheld devices for point-of-care testing. For instance, biosensors are found in instruments measuring glucose, drugs of abuse, pregnancy and many more.
Due to the direct response of a biosensor to its analyte, they are often used in real-time monitoring of a molecule or a process connected with it. Such an analysis not only gives quantitative information about the presence of a molecule but adds the temporal component of analyte presence.
What are biosensors used for?
The few examples of biosensors that were already mentioned indicate it: the areas in which they are used are numerous and diverse. Following, fields and typical sensors will be presented.
Biosensors in food technology and safety
The food industry applies biosensors in order to assess product safety, namely microbial contaminants, pesticides and toxins or to monitor product quality by detecting specific food components. One example for biomolecule-based assessment of toxins in food is the aforementioned microbial arsenic biosensor. It reports the presence of harmful arsenic by using the genetic regulation of a resistance mechanism that in presence of the hazard instead of an arsenic transporter system expresses GFP.
An example of a biosensor determining the quality of a food is the glutamate sensor: glutamate enhances flavor and is found in natural products but is added particularly to highly processed food products. The glutamate detection is often based on the enzyme glutamate oxidase that specifically converts glutamate to alpha-ketoglutarate. Similar to a glucometer, the glutamate oxidase catalyzed reaction is detected within the biosensor by a change in current which directly reports on the presence of the flavor enhancer glutamate.
Environmental monitoring by biosensors
Biosensors also test environmental samples or samples released into the environment for pollutants. A commonly used device measures the biochemical oxygen demand (BOD) of water or, more specifically, microorganisms living therein. The BOD is important to estimate the organic resources of a water sample that can be used by microorganisms to grow and hence the likeliness to be polluted with organisms. It is typically measured in water designated to be discharged. The commercially available biosensor measures BOD with a combination of microbes consuming oxygen in the presence of organic compounds and a Clark electrode that determines oxygen concentration.
Recently, novel biosensors were developed that detect residual amounts of pharmaceuticals in wastewater of municipal treatment plants. This is of particular importance to prevent excessive release of polluted water into the environment and exposure of water organisms to drugs. The two most commonly addressed drug targets can monitored by the cell-based sensors: cyclooxygenase-1 (COX) that is inhibited by common pain killers and beta-adrenergic receptor that is blocked by drugs against hypertension and arrhythmias. The COX-inhibitor sensing cell line is based on CHOs that carry a fluorescent ratiometric biosensor. A detailed description of the assay principle is found in AN322: Cell-based assay detects residual nonsteroidal anti-inflammatory drugs (NSAIDs) in effluent of municipal wastewater treatment.
The second cell-based biosensor detects β-blockers by means of cAMP production. The second messenger molecule is produced upon stimulation of the drug target, the beta-adrenergic receptor (Fig. 2). Activation of the receptor is prevented, for example, by β-blockers like Metoprolol. However, in reponse to β-blockers, no cAMP is produced and the divergence can be monitored with a FRET-based cAMP senor called CEPAC. AN319: Cell-based assay detects residual β-blocker substances in effluent of municipal wastewater treatment plants explains the principle of the CEPAC senor and how it was used to report on residual drugs in wastewater.
The production of cAMP linked to the activation of the receptor can be monitored with a FRET-based biosensor called CEPAC (Fig. 3). AN319: Cell-based assay detects residual β-blocker substances in effluent of municipal wastewater treatment plants explains the principle of the CEPAC senor and how it was used to report on residual drugs in wastewater.
Medical biosensor applications
The handheld glucose measurement device was mentioned earlier. It helps diabetics to monitor their glucose levels at home and prevent hyperglycemia. Another emerging biosensor application indicates acute myocardial infarction, a major cause of death. Several systems are commercially available that all detect cardiac troponin, a protein complex indicating heart muscle damage. The biosensor uses an anti-troponin antibody as biological component which binds specifically to cardiac troponin. A secondary antibody subsequently binds to the captured analyte and acts as a mediator in changing the current in the presence of troponin. The biosensor system is integrated into a handheld device and is used by clinicians to diagnose acute myocardial infarcts.
Applications and development of biosensors in life science research
Nothing stops the fast and direct analyses with the help of biosensors to benefit life science as well. As the needs in research laboratories are diverse, a lot of effort is made in developing biomolecule-based sensors. These can often be genetically encoded within a cell line of interest and therefore report on changes of a biomolecule in real-time, in the cell of interest, and even in the cell compartment of interest. A typical principle is the use of a modified fluorescent protein such as green fluorescent protein (GFP) that is linked to a binding domain specific for the analyte. Association of the analyte with the fluorescent biosensor results in a conformational change and subsequently in an increase in fluorescence that can be detected with a microplate reader. Another fluorescence-based biosensor principle uses Förster resonance energy transfer (FRET) between two fluorophores in the vicinity. Again, a protein domain binding specifically to the analyte enables detection. The protein is further labelled with two fluorophores which can transfer light from one to another only when they are proximate. The proximity is induced by a conformational change upon analyte binding and is read out by measuring both fluorophores in a microplate reader.
An example of a fluorescent biosensor is reduction/oxidation sensitive GFP (roGFP). It contains additional cysteines that form a disulfide bond when oxidized. The oxidized and reduced form of roGFP display different optical characteristics (excitation spectra) which can be measured in sensitive microplate readers. The roGFP sensor is typically expressed by cells and reports in real-time on their redox state as well as on the redox state of specific proteins and enzymes. Mostly, the cells carrying the biosensor are exposed to various treatments and are analyzed in microplate format to enable the required throughput. Learn in the video below how these sensors are used and why sensitive biosensor detection is needed.
In comparison to changes in redox state, the responses occurring upon activation of G-protein coupled receptors (GPCRs) are quicker, but just as well measurable with biosensors. GPCRs are the most abundant drug targets and therefore under continuous research. One response to activation of a GPCR coupled to an intracellular Gq subunit is the release of calcium ions (Ca2+) and diacylglycerol (DAG). With the help of two fluorescent biosensors with differing spectral properties, both second messengers were detected in the real-time and cellular background using the CLARIOstar microplate reader. The benefits of biosensors reporting on GPCR are explained in the video below.
In the following example another second messenger associated to GPCR activation was determined: cAMP. The levels of cAMP increase upon activation with synthetic cannabinoid receptor agonists was monitored with a BRET-based biosensor named CAYMEL.
