Developing an optically stimulated piezofilm immunoassay

The point-of-care (POC) or near-patient test market remains a strong growth area in the IVD industry.¹ Two of the main factors driving growth in diagnostic POC testing are convenience and a compelling need to know.

In today's digital age, society has become more impatient, demanding information as soon as possible. Consequently, patients can walk into a pharmacy or physician's office and have their cholesterol levels measured while they wait.

However, the role of POC testing for acute diseases can be critical. If a patient presents at an emergency room with chest pains, it is more than just convenient to have a rapid measurement of troponin, CKMB, and myoglobin levels to aid in diagnosing a potential heart attack.

Centralized laboratory testing is characterized by high throughput and low costs. In order to compete with the labs, POC tests need to be inexpensive and user-friendly, and they must match the performance of lab tests. Ease of use is also critical for obtaining CLIA-waived status, which is an FDA approval that allows a test to be conducted by nonexpert users. This waiver means that a test needs to “employ methodologies that are so simple and accurate as to render the likelihood of erroneous results negligible; or pose no reasonable risk of harm to the patient if the test is performed incorrectly.”² Two examples of technologies that normally meet such criteria are lateral-flow tests and electrochemical enzymatic tests.

Lateral-flow tests, or immunochromatographic strips, were first introduced as qualitative urine pregnancy tests used in doctors' offices and at home. They have evolved into rapid tests for a range of analytes, including HIV, respiratory diseases, drugs-of-abuse, cardiac markers, and infectious diseases. Although their use has become widespread, lateral-flow tests have sensitivity limitations and tend to be semiquantitative. Recent improvements in lateral-flow testing have been made using both fluorescent and magnetic labels.³

Electrochemical enzymatic tests were developed primarily for measuring the glucose levels of diabetic patients. This technology has also evolved, and electrochemical tests are available for coagulation and immunoassays, such as the i-Stat test by Abbott Point-of-Care (East Windsor, NJ) for troponin I.^4,5

Other POC testing technologies exist, including variants on lateral-flow. For example, the Triage test by Biosite Inc. (San Diego) uses a capillary channel rather than a capillary membrane.⁶ While many technologies have shown promise as alternatives for improved POC testing, they have not yet realized their potential (e.g., lab-on-a-chip formats). There are various reasons why such technologies have fallen short, such as performance, cost, complexity, and manufacturability. Integrating complex optics into an instrument or a disposable can dramatically increase costs.

Other complications include using additional onboard reagents, the need for wash steps to remove excess reporters, and separating red cells from the sample. POC tests should ideally use fingerstick samples of capillary blood, which limits the sample volume to approximately 30 microliters. While this amount is more than adequate for a glucose test, separating plasma from cells in a 30- ml sample without significant sample loss is challenging. Adding and trying to find a solution to this complexity can quickly turn a potential POC test into expensive laboratory equipment.

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Table I . (click to enlarge [5]) Key requirements for a quantitative POC immunoassay test.

The next generation of POC devices will need to meet a number of key requirements (see Table I [5]). Some requirements are more critical than others and will vary depending on the analyte, such as detection limit, dynamic range, and precision. However, in order to use a fingerstick blood sample in POC tests, the sample volume requirements should be minimized and the test should be conducted with whole blood. If a single technology could fulfill all of the requirements in Table I [5], it could be used in a range of diagnostic applications. This article discusses a novel technology that could meet the requirements in Table I [5].

Piezoelectric Properties

For centuries, generating an electric charge in certain crystals (e.g., quartz) through either mechanical strain (piezoelectric effect) or heating (pyroelectric effect) has been a well-known phenomenon. The earliest mention of the pyroelectric effect was in 314 bc by the Greek philosopher Theophrastus, who observed that tourmaline becomes charged when heated.⁷ Such effects rely on crystal structure asymmetries, leading to an alignment of the dipoles throughout the crystal. When the crystal is stressed, these dipoles generate a charge.⁸

The piezoelectric effect can be either direct (strain generates electrical polarization) or inverse (an electrical field generates a mechanical effect). Unsurprisingly, this effect has many useful applications, and piezoelectric sensors and actuators are used in various devices, including microphones, compact disc players, accelerometers, quartz crystal microbalances, and pressure monitors. Perhaps the most common application is found in electric lighters, in which butane gas is ignited from the spark generated by squeezing a quartz crystal.

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Figure 1. (click to enlarge [6]) The strong dipoles due to the carbon-fluorine bonds in PVDF are responsible for the high piezo- and pyroelectric coefficients.

Some polymer films can also be manufactured with piezoelectric properties by exposing them to an electric field during the manufacturing process.⁹ The electric field aligns the dipole moments in the polymer, which creates the necessary asymmetry in structure. Such polymer films have several advantages over crystalline materials, including cost, robustness, and flexibility. The most commonly used piezoelectric polymer is poled polyvinylidene fluoride (PVDF), which exhibits a piezoelectric charge constant that is 10 times greater than quartz (see Figure 1 [6]).

Piezo-Optical Sensors

A simple example of a piezo/pyro-optical sensor is an infrared motion detector that can trigger external lights to turn on automatically at night. Heat dissipation from a human or an animal causes localized heating of a pyroelectric element in the sensor, which triggers an electrical signal and signals the external light to be turned on.

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Figure 2. (click to enlarge [7]) The sensor system. Light is pulsed from an LED source and absorbed by a colored material on the piezofilm surface (shown in black). Localized heating causes stress in the film, generating a voltage, which is amplified and fed into the instrument.

Similarly, if visible light is projected on a colored material on the surface of a piezofilm, an electrical signal will also be generated.¹⁰ Upon absorption, the light energy is converted into heat and is dissipated into the sensor, causing thermal and mechanical stress which is manifested as a voltage. This method can quantitate color changes on a surface or changes in optical properties (see Figure 2 [7]). The light-emitting diode (LED) illumination can be directed through the thickness of the piezofilm, ensuring that the light intensity is maximized at the surface of the film in case of an opaque, translucent, or dirty sample.

Applying Piezo-Optical Technology to Immunoassays

A standard immunometric or sandwich assay needs to be able to discriminate between the fraction of labeled antibody that is bound to the analyte of interest and the fraction that is unbound. Such discrimination is achieved by using solid-phase capture surfaces in combination with wash steps, although other methods (e.g., time-resolved fluorescence) are also available. By attaching capture antibody to the surface of a piezofilm, an assay can discriminate between the labeled antibody bound to the surface of the film and the antibody in solution without conducting a separation step. The labeled antibody generates localized heating upon illumination.

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Figure 3. (click to enlarge [8]) The stress generated in the piezofilm from components near the surface swamps the signal from components in the bulk.

However, the degree of stress in the film depends on the distance of the labeled antibody from the film. If the labeled antibody is far from the film's surface, most of the heat dissipates into the bulk medium. If the labeled antibody is close to the film's surface, more heat will dissipate into the film and more charge will be generated. In addition, a key benefit of the piezo-optical technology is that other absorbing components in the system, particularly red cells, give minimum signal from the bulk sample (see Figure 3 [8]). Further differentiation between surface and bulk signal is enhanced by the piezofilm's time response with respect to the pulse of light. Materials close to the surface transfer heat more rapidly to the piezofilm.

Of course, other methods can distinguish between events distant from a sensor surface, particularly surface plasmon resonance and surface acoustic wave techniques. One advantage of surface plasmon resonance techniques is that no label is required on the binding material. However, surface plasmon resonance requires complex optics and is used more as a research tool. Advantages of the piezo-optical approach include the low cost and simplicity of the components, which make such a system attractive for POC immunoassays.

Although the piezo-optical method is not electrochemical, it has some similarities with electrochemical tests. For example, the piezo-optical approach elicits a direct electrical output from the film, unlike methods such as fluorescence or luminescence in which an optical signal needs an intermediate conversion step into an electrical signal. This direct electrical output keeps the system simple.

Label Choice

In developing a piezo-optical immunoassay system, choosing the proper label is critical. The label is responsible for absorbing the light energy and transferring the heat generated to the piezofilm. Among its key attributes, the label should have a high optical density to maximize the amount of light that is absorbed, be readily conjugated to antibodies, and be an appropriate size such that diffusion to the piezofilm surface is rapid. In order to minimize any interference from red blood cells, the ideal illumination wavelength is 650–700 nm.

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Figure 4. (click to enlarge [9]) Absorbance profile of oxygenated (blue) and deoxygenated (red) hemoglobin. Oxygenated hemoglobin is predominant in healthy human capillary blood samples.

At this wavelength, light absorption by oxygenated hemoglobin is at a minimum (see Figure 4 [9]).

Colored antibody conjugates for immunoassays are well known, particularly in lateral-flow strips which widely use latex and gold colloidal particles. However, carbon colloid works particularly well in a piezo-optical system. The optimum particle size is around 150 nm, which is large enough to generate a strong signal, but not so large that the diffusion rate in solution becomes prohibitively low. Antibodies are readily conjugated to the surface of the carbon particles by passive adsorption.¹¹

Instrumentation and Theoretical Considerations

In order to be able to pick up an electrical signal, the piezofilm's surface needs to be coated in a transparent conductor, such as indium tin oxide, a common component in LCD displays. The piezo-optical system does not require complex optics, and the light source can simply be an LED which is flashed on and off. The pulsing of the LED is needed because the signal is generated as a timed response, meaning that the piezofilm responds to changes in stimuli. Once the electrical output is fed into a locked-in charge amplifier, it is converted into an arbitrary digital signal (counts) via an analog-to-digital convertor (ADC). The counts value depends on the maximum signal that is generated in the system, combined with the ADC's resolution. The piezo-optical system's electronic components are not any more complex than a handheld glucose meter.

The actual process of heat generation in different solid and liquid layers, which is followed by transfer through the different components of the piezo-optical system over time, is complex. While a theoretical model is currently being developed, empirical processes have been used to optimize the film's response, the flash rate of the LEDs, and processing of the signal to obtain maximum signal-to-noise ratio.

A frequently asked question is, “How do vibration, noise, and thermal drift affect the signal from the film?” While these factors can affect the signal from the film, several preventative measures can eliminate or drastically reduce any interference. Such measures include mechanical isolation, band-pass filtering, common-mode noise rejection, and software processing. By implementing such measures, the instrumentation's sensitivity is not limited by any external factors.

Assay Principles

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Figure 5. (click to enlarge [10]) Kinetic profile of carbon label binding to piezofilm surface in the presence of 5 µIU/ml (800 pg/ml) TSH.

In an immunometric assay in its simplest form, a piezoelectric film is applied as follows. A capture antibody is coated on the piezofilm's surface. A carbon-labeled antibody is resuspended with the sample and presented to the film's surface. Sandwich formation of the capture antibody, analyte, and labeled antibody at the film's surface takes place over time under diffusion control. No active mixing or washing occurs during the measurement process. During this period, kinetic measurements of signal growth are taken (see Figure 5 [10]). In this figure, four measurements are shown, two signals and two negative controls. Each sample (40 µl of serum) contains human thyroid-stimulating hormone (TSH) at 5 µIU/ml (approx. 800 pg/ml or 2.6 × 10^-11 mol dm^-3), and the negative controls have TSH levels of less than 0.01 µIU/ml.

A key aspect of the piezo-optical system is that continuous measurements are taken during the binding process. Once enough data points have been established to give a confident estimate of the slope, the results can be generated. For high analyte concentrations, such results can be produced rapidly. In Figure 5 [10], the slope can be accurately determined in only one or two minutes, which does not require complex algorithms. Standard statistical methods can carry out a regression analysis and calculate a confidence interval for the slope.¹² In this example, the protein concentration is approximately 800 pg/ml in a 40-µl sample. In addition, taking kinetic measurements helps to give a wide dynamic range at the top end because signal analysis is carried out prior to reaching saturation.

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Figure 6. (click to enlarge [11]) A calibration curve of a carbon assay for TSH.

By running such kinetic profiles at different analyte concentrations, a calibration curve can be generated (see Figure 6 [11]). In this figure, the data are extrapolated from kinetic measurements taken during a 10-minute period at each TSH concentration.

A good linear relationship exists between concentration and signal, with good precision. The coefficient of variation at 1 ng/ml in human plasma is less than 5%, and the system's analytical sensitivity has been proven down to 22 pg/ml.

As stated earlier, the piezo-optical system can take measurements in dirty samples without any wash steps. Red blood cells are the main interference, due to the high concentrations of hemoglobin (approximately 140 g/L) in blood. However, such interference can be minimized by illuminating the sample at 650 nm, at which any light absorption is minimized. Developing high-powered LEDs at this wavelength has been driven by lighting applications, particularly in the automotive industry. Proprietary methods have also been developed for the piezo-optical system to minimize red cell interference, which are currently being implemented in the system. While further work on whole blood is required, the data are very encouraging.

Commercialization

Figure 7. The instrument being developed by Vivacta.

The piezo-optical detection system is being commercialized as a POC system by Vivacta Ltd. (Kent, UK), a venture-capital-funded start-up company. The initial focus is to develop a professional device for use in doctors' offices. Prototype test-bed instruments and cartridges have been developed for lab evaluation. In collaboration with a number of partners, including Integrated Technologies Ltd. (Kent, UK), a commercial reader system that can work with disposables is also being developed (see Figure 7).

For these systems, TSH has been chosen as the initial test analyte for a number of reasons. First, TSH is an excellent stand-alone analyte for screening and monitoring thyroid function. Second, TSH has a demanding detection limit and wide dynamic range, which would demonstrate the technology's potential and prove its capabilities for use with alternative analytes, such as cardiac markers.

In addition, a disposable piezo-optical device is being developed in parallel with the instrumentation. This disposable device will house the piezofilm and its electrical contacts. It will also contain dried-down reagents and some elementary fluidics to resuspend the reagents with the sample and present the mix to the piezofilm surface. Two alternative routes are being explored for the disposable device construction. The first route uses an injection-molded core, to which the piezofilm is attached with a pressure-sensitive adhesive. The second uses simple lamination processes, similar to glucose strip fabrication.

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Steve Ross, PhD, is director of R&D at Vivacta Ltd. (Kent, UK). He can be reached at sar@vivacta.com [12].

Other Applications

Although TSH has been chosen as the analyte to take the piezo-optical technology to market, other potential application areas exist. Such areas include competitive assays, nucleic acid assays, and multiplexing applications. Multiplexing is a particularly attractive application, because only one electrical connection needs to be made to the film to measure several analytes. By addressing different capture areas with light sequentially and interrogating the signal over time, multiple analytes can be measured simultaneously. Multiplexing is ideal for cardiac markers, in which a diagnosis is rarely made based on the results of one test.

Conclusion

The piezo-optical technique described has great potential for POC diagnostics. With the simplicity and low cost of the instrumentation and the sensor, along with its ability to detect in real time with no wash steps, the piezo-optical technology offers several benefits compared with other competitive technologies.