BIOMEDICAL SENSING

CHRISTOPHERS. BRAZEL University of Alabama Tuscaloosa, Alabama

INTRODUCTION

In recent years, biomedical diagnostics research has led to the development of simple, less invasive, and more accurate evaluative techniques. Much of this is due to biosensor technology, which has played an important role in material improvements used to sense, carry signals, and respond to signals. Biosensor devices can measure micromolar or even smaller quantities of biological substances, including chlorides, glucose, lactose, and urea. Pietro (1) defines biosensors as “any discrete sensing device that relies on a biologically derived component as an integral part of its detection mechanism,” although sensors that are used to monitor biological conditions are usually also included.

Medical applications abound, ranging from diagnostic tests for home testing toin vivomonitoring of vital conditions and using feedback to control drug delivery or send am- plified signals of a change in patient health. Smart materials are frequently combined with biological components to create systems that respond to environmental conditions, such as temperature, pH, concentration of particular analytes, or even light. These smart materials are typically polymeric, and most have the common characteristic that changing environmental condition cause a thermody- namic change between hydrophilic and hydrophobic states, as detailed later. pH paper strips may be one of the sim- plest sensing devices; they relay a physiological condition through a colorimetric response, and because they are a form of dry chemistry, they simplify detection and diagnostics, which is especially important for home diagnostic kits.

This article presents the use of intelligent polymers in sensing systems, that range from traditional biosensors to advanced self-responsive systems. This review of biomedical diagnostics using smart polymers examines some of the medical applications for intelligent polymers used in electrode-based systems and in bioconjugate systems.

• • • •

• •

•

• Drug

S S

E P PE

P H⁺

H⁺ H⁺

Sensing Signal Response Drug delivery

transmission

E E

··

· ··

Figure 1. Steps involved in biomedical sensing using intelligent polymers. First, the substrate (S) diffuses to an enzyme immobilized in the polymeric device and reacts; the signal that a particular substrate is present is then transmitted to the surrounding polymer by diffusion of the products (P, H⁺) of the enzymatic reaction, such as hydrogen ions. The hydrogen ions, in turn, ellicit a swelling response from a smart material, such as pH-sensitive poly(acrylic acid), which can then deliver an embedded drug by diffusion.

Sensing devices are categorized by the mechanisms of signal detection and response, and examples of systems that monitor analytes, such as glucose, are addressed. Addi- tional modes of response beyond the traditional electronic signal as well as synthesis techniques and examples of medical diagnostics using smart polymers, will be discussed.

Smart materials sense their environment, judge the magnitude of changes, and respond to obtain the most ther- modynamically favorable state. This response can cause changes such as surface modification (from hydrophobic to hydrophilic or vice versa), swelling or shrinking of gels, enzyme solubility (if covalently attached to a phase- separating polymer), and binding of polymer and proteins (especially in reversible ionic interactions) (2). These reversible phenomena are useful for biosensors in protecting biological materials, collecting and concentrating analytes, and responding to stimuli to transduce a signal or deliver a drug (3). Smart materials act as on/off switches and can be barriers for an enzyme to protect it from the body’s immune system and from harmful solutes or pH conditions. The ability of polymers to phase separate can also aid in separating and preserving enzymes or antibodies to be reused in future diagnostic tests.

The use of intelligent polymer systems in sensors provides the possibilities of combining sensing, transduction of signals, and response in the same independent device and the possibility of controlling biological events based on the signal by delivery of drugs or other means. These three behaviors (sensing, transduction, and response) are characteristic of biosensors, although the traditional response is often electronic (Fig. 1).

Sensing and response processes in smart materials are reversible, and after the hydrogen ions diffuse from the polymer, it returns to its normal unswollen state, and drug release stops. This creates a positive feedback mechanism whereby the device senses abnormal biological or biomedical events and treats the diseased state only when the triggering molecule is present. An example of the structure of a feedback drug delivery biosensor is shown in Fig. 2, where a silicon chip is used as a platform for the biosensor (4). This is similar to the current research of the National Science Foundation’s “Lab on a Chip” technology program, where chemical moieties are analyzed on a molecular scale. In the scheme shown, drug reservoirs are kept

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Poly(acrylic acid) molecular gate Drug reservoir Gate: φ 150 × 400 µ m

Si water 5 mm

5 mm 1.5 mm

Drug

Pyrex glass Pitch Chip

Figure 2. Model scheme for the structure of a sensor device [reprinted with permission from (4);

behind molecular gates made of pH-sensitive poly(acrylic acid) to yield a sensor that provides a drug delivery feedback response to changing pH.

Traditional biosensors include electrochemical sensors that have been under development since the early 1970s and in which enzymes, antibodies, chemoreceptors, and cellular tissue are used as catalysts for reactions that create electrical signals (5). An example of an electrochemical sensor is shown in Fig. 3, where an enzyme is deposited directly onto a metal conducting electrode and a polymer or protein is used to immobilize the enzyme. This

Figure 3. SEM photograph (200x) of a portion of the working electrode that had glucose oxidase and albumin electrodeposited and cross-linked onto the exposed surface. The visible metal layer surrounding the layer of glucose oxidase and albumin is the con- ductor underlying the insulating layer [reprinted with permission from (6); copyright 1994 American Chemical Society].

particular device was designed for subcutaneous implan- tation to monitor glucose concentrations in diabetic patients (6). The magnitude of the sensor response is proportional to the analyte concentrations, but the response also depends on the diffusion of the analyte to the enzyme or receptor, the kinetics of the enzyme reaction, and the diffusion of reaction products (such as O2 or H2O2) to an electrode. Biosensor technologies include biochemical sensors, enzymatic sensors, cellular sensors, sensors for redox reactions, antigen/antibody interactions, and other materials that provide recognition surfaces (7). They typically include a specific reaction site (sensor), a mode of trans- porting the signal (transducer), and a signal measurement or feedback mecanism (responder). These sensing materials can involve antibody/antigen (8) or enzyme/substrate interactions, where the interaction is highly specific and a singular target analyte can be recognized (9). Organelles, whole cells, or tissue can also be used as the sensor (10).

Spichiger-Keller (11) divides the types of sensors by the recognition processes used (Table 1). Biosensor devices are one class of intelligent materials, but they can also be combined with smart polymers (such as those sensitive to environmental pH or temperature) to aid in signal collec- tion through specific interactions with the analyte, signal transduction, or the response mechanism (as depicted in Fig. 1).

Because of the complex nature of biosensors, it is important to understand the requirements of sensing in general, and then apply those guidelines to the additional requirements needed to use the sensor in a biological environment.

Complications can arise when multiple functional materials are combined into the sensor device. Several important characteristics of biosensors ensure accurate, reproducible, and specific results. Diamond (12) summarized some of the important considerations in Table 2. Many of the characteristics, such as reproducibility of results, robustness, and proportional signal output, are common to process control theory. In addition to the requirements of Table 2, materials (especially the surfaces exposed to biological environments) must be biocompatible, the device should be as noninvasive as possible for medical applications,

BIOMEDICAL SENSING 97

Table 1. Classification According to the Type of Recognition Process^a Chemical Reaction

Sensor Type Reacting Pairs Recognition Process

Chemical sensors in the Host–guest; ligand–analyte; Complexation

strict sense carrier–ion; association, addition,

ion, neutral species, typical equilibrium

and gas sensors reactions

Oxide semiconductor Inorganic metal oxide; Absorption, reduction,

sensors layer–reactive gases oxidation

Enzymatic sensors Active site–substrate– Metabolic turnover, cosubstrate mediated sensing typical steady state, reactions; active site–

mediator-electrode Kinetic reactions Immunochemical sensors Antibody (catalytic antibody)– Affinity, association,

antigen; antibody–antigenic

protein–hapten Equilibrium reactions Receptrodes, Receptor–substrate Association, affinity,

living organs, bilayers metabolic turnover

hybrides as abzymes, etc.

Table 2. Ideal Characteristics of a Sensor^a

Characteristic Comments

Signal output should be proportional This is becoming less important because or bear a simple mathematical of on-device electronics and

relationship to the amount of the integration of complex signal processing species in the sample options to produce so-called smart sensors.

No hysteresis The sensor signal should return to baseline after responding to the analyte.

Fast response times Slow response times arising from multiple sensing membranes or sluggish exchange kinetics can seriously limit the range of possible application and prevent use in real-time monitoring situations.

Good signal-to-noise(S/N) The S/N ratio determines the limit of characteristics detection; can be improved by using the

sensor in flow analysis rather

than for steady-state measurements; S/N ratio can also be improved by filter or impedance conversion circuitry built into the device(“smart” sensor).

Selective Without adequate selectivity, the user can not confidently relate the signal obtained to the target species concentration.

Sensitive Sensitivityis defined as the change in signal per unit change in concentration (i.e., the slope of the calibration curve); this determines the ability of the device to discriminate accurately and precisely between

small differences in analyte concentration.

a(From (12) copyright^C 1998. Reprinted by permission of John Wiley & Sons, Inc.

98 BIOMEDICAL SENSING

and biosensors must have adequate lifetimes, especially if implanted. Immobilization is often necessary in enzymatic or immunosensors, so that the active material can be kept near the electrode or other transduction and feedback device.

The ideal biosensor characteristics listed in Table 2 provide some of the driving forces for research in this field.

Hysteresis limits the effective lifetime of a sensing device;

because of repetitive cycling, the sensor’s response becomes less reproducible. Poshossian et al. (13) report a penicillin biosensor based on a pH-sensitive gel that has a hysteresis of less than 4 mV (less than 0.4% of the signal) and a usable lifetime of at least one year; perhaps more im- portantly, they demonstrated that by using an absorptive technique to immobilize penicillinase, the sensor could be regenerated by desorption and resorption of fresh enzyme.

To achieve faster responses in immobilized enzyme-based systems, macroporous gels have been employed to reduce any potential hindrances due to diffusion (14). The limits of sensitivity are also important in creating sensing devices.

The minimum detection level has dropped as low as the nanomolar level (15), but the range of analyte concentration across which the biosensor is useful must also match the system being monitored.

Although the functioning of a biosensor must be accurate and robust, the largest barrier to successful imple- mentation of a biomedical sensing device is ensuring that it is readily usable and simple for patient compliance. The development of assays that extract metabolites across the skin (16–18) or those that sample biological fluids that are more readily collected, such as urine, sweat, or saliva (19), are examples.

Compared to all sensor markets, the medical and biological fields have become leading drivers for new research in recent years and have great potential for improving pharmaceutical processing, medical diagnostics, and patient treatment (12). The driving forces for future development of biomedical sensing devices include improving and diversifying recognition mechanisms, developing new materials for immobilization that meet the stringent requirements of biocompatibility, developing materials that do not use enzymes or biological components (using techniques such as enzyme mimics and molecular imprinting), improving the flexibility of design (especially through dry chemistry and removing the requirement for electrolytic fluids in the sensor), discovery and development of new sensor molecules that are highly specific to diseased states, and improving signal processing and reproducibility (12).

Many biosensors have a linear response to concentrations across only a narrow window, so one continuing focus will be to develop sensors that are more robust and have proportional responses across a wider range of analyte concentrations.

A variety of biological components have been used in sensing mechanisms for biosensors; the majority is based on enzymes because enzymatic reactions are highly specific, occur at low analyte concentrations, and can differ- entiate between enantiomers and compounds of similar structures. Particular enzymes can also be selected to cat- alyze any of a range of medically significant biochemical reactions. Because obtaining highly purified enzymes or

proteins is often expensive, McCormack et al. (9) suggest using whole cells in the substrate recognition step. Cell- based sensors would be more adaptable and resilient compared to proteins or enzymes and may lead to biosensor products that have longer lifetimes; cells can carry out more complex reactions by using multiple enzymes and metabolic pathways to produce a product that signals a response from the biosensor. On the negative side, cell-based biosensors are much more complex, and the direct cause–

effect (reactant–product) relationship of enzymatic reactions becomes more difficult to define. One example where a cell-based sensor would be preferred is in monitoring the products of cell metabolism to determine the availability of nutrient supply. If carbon or nitrogen sources are in short supply, secondary metabolites may be formed, which could be monitored; alternatively, if the oxygen supply is short, partial metabolites, such as lactates, may build up in the tissue near the biosensor. This would be potentially useful in monitoring cells used for tissue engineering or implanted organs to verify that the region is becoming vas- cularized and not rejected by the host. It is also possible to use multiple cells or even plant or mammalian tissue in combination with sensors to monitor the production of highly specific metabolites.

MEDICAL, THERAPEUTIC, AND DIAGNOSTIC APPLICATIONS OF BIOSENSORS

Biosensors have made it possible to reduce human health care costs by using available at-home test kits so that patients can monitor their own glucose levels, pregnancy hormones, and cholesterol (20). Most of these kits can monitor easily collected biological specimens, such as saliva or urine, or require training the patient to collect blood samples (for glucose monitoring). Several ex vivo diagnostic tests, including glucose and cholesterol screenings, have been made possible through biosensors that incorporate enzyme assays into dry chemistry electrodes (21). In many of these tests, polymers are included with the biological sensing component along with a simple readout, such as an indicating dye. Biosensors are used in many biological systems, including control of reactions in biological reac- tors for producing pharmaceutical agents (22), measuring analytes in biological samples, such as blood or urine (23), and monitoring health informationin vivo(16).

As an example of biosensors used with smart materials, Mizutani et al. (24) developed enzyme electrodes based on oxidoreductase enzymes for monitoring lactates and ethanol. Their device consisted of a stimuli-responsive

“smart” polymer system of poly(L-lysine)/poly(4-styrene- sulfonate) to which enzymes are bound by ionic interactions with the polymers. The ionic polymer is involved in both enzyme immobilization and in screening solutes such asL-ascorbic acid and uric acid, which interfere with signal transduction if they are near the electrode. Lactate levels in sour milk and human serum were tested using the biosensor and were compared to conventional test kit methods (Table 3). Results must compare favorably in ac- curacy and reproducibility to consider biosensor devices feasible.

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Table 3. Comparison of Results Obtained forL-Lactic Acid in Human Sera and Sour Milk by Different Methods^a

L-Lactate Concentration (mM)

Sample Proposed Method F-kit method

Serum 1 2.03 2.02

Serum 2 1.44 1.36

Serum 3 2.80 2.86

Serum 4 1.75 1.67

Sour milk 1 63.8 61.4

Sour milk 2 74.6 73.0

Sour milk 3 53.3 53.6

Sour milk 4 87.7 86.1

Sour milk 5 72.4 74.4

Medical applications of biosensors cover a wide range of analytes (Table 4) and medical conditions (Table 5).

Diabetes monitoring and treatment is the primary thrust of research and product development in current medical sensing technology, but as the understanding of molecular biochemistry advances, there are possibilities of developing economically feasible sensors based any of these conditions and more.

Some of the enzymes of potential use in biomedical sensors are listed in Table 6. Much of biosensor development has focused on oxidoreductases because changes in oxidized states of chemicals cause electron flow that can be detected by using electrodes.

Biomaterials that respond to environmental changes and fractures to self-repair are considered smart materials that sense biological events and give feedback by releasing healing chemicals. In these systems, the “sensing” is not as sophisticated as in traditional biosensors, but the use of self-repairing structures in biomaterials makes them able to sense stresses and cracks and respondin vivowith- out surgical procedures to replace or reset the implant.

Dry (30) described techniques to improve biomaterial per- formance by using smart materials that self-heal upon mechanical erosion. This is particularly important in developing load-bearing biomechanical materials for replac- ing bones and joints. Hastings (33) also cites the use of smart materials that self-repair upon shear and fracture or release drugs or hormones in conjunction with biomaterial implants to reduce inflammatory response. These devices typically use encapsulation to hold the active ingredient, and shear or pressure are used to break the capsule wall and trigger release.

Another area where biosensing is done nontraditionally is targeted drug delivery. Some of the same interactions, especially using chemoreceptors, can be used to design drug

Table 4. Examples of Chemical Analytes Subject to Biosensing

Lactate Pyruvate Glucose

Fructose Galactose CO₂

O₂ Ascorbic acid Cholesterol

Urea/uric acid

Table 5. Examples of Biomedical Applications of Sensing Materials

Diabetes monitoring and treatment (25) Detection of viruses/toxins (26, 27)

Monitoring metabolic substrate use (biological oxygen demand) (9)

Monitoring metabolic products (such as lactates or partial oxidation metabolites) (28)

Determining the efficiency of dialysis and filtering (in organs orex vivo) (19)

Biopharmaceutical production and testing (e.g., monitoring cell activity in fermenters) (22, 29)

Repair of fractured tissue or bone (30)

Targeted drug delivery (such as cardiovascular or gastrointestinal) (31, 32)

carrier surfaces so that a drug is delivered to a specific area.

Yang and Robinson (34) used glycoproteins to bind drug delivery vehicles to a selected site. These surface deriva- tives may be used to anchor controlled release devices or biosensors to a particular type of cell in the body. Smart polymers used in biosensors for drug delivery are detailed later in this article.

Monitoring lactate and pyruvate levels by using a biosensor can indicate when secondary and partial oxidation products are formed in vivo and indicate when the supply of nutrients or oxygen to tissue is insuffi- cient (35). These enzymatic electrochemical sensors are used in extracorporeal evaluation of blood in patients who have an artificial pancreases; and the lifetime of the sensor is 30 days/300 assays. The results from the biosensor correlated well with spectrophotometric analysis of blood serum.

Many biomedical sensing devices have been proposed and constructed for monitoring blood glucose levels, and some of these approaches are detailed later in this article.

The reader is referred to Campanella and Tomassetti (36) for a review of biosensors for clinical and pharmaceutical analysis.

POLYMERS AS ELECTRODE COATINGS AND BIOSENSOR MEDIATORS

To date, most research on biosensors has focused on devices that consist of a metal layer to conduct electrons as the signal, an enzyme or antibody to sense the presence of a particular analyte, and a membrane to immobilize the enzyme and also possibly aid in transducing the signal to the electrode. A common approach used to design glucose sensors, as demonstrated by Johnson et al. (6), is based on glucose oxidase, which is immobilized between an outer membrane permeable to glucose and oxygen and a platinum electrode to reduce the formed hydrogen per- oxide and transmit electronic signals (Fig. 4). The design of the membrane is crucial to operation because the pore size must allow rapid diffusion of glucose and oxygen and yet retain the enzyme.

Polymer gel coatings on electrodes serve multiple pur- poses. Polymer gels form semipermeable membranes to

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