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Detection Technologies

While the consumer IVD market has undergone tremendous growth in recent years, it has been primarily dominated by glucose monitoring tests. A limited number of other types of IVD products are available on the over-the-counter (OTC) consumer market, and many of them can generate only qualitative test results for a small number of conditions. In order to expand the OTC market, the desired IVD products in this market need to be inexpensive but yet should deliver quantitative results. For example, C-reactive protein (CRP) tests for cardiac risk assessment need to provide accurate measurements in a low concentration range. The ultimate goal for Kimberly-Clark (Dallas) is to develop a general assay platform that consists of a low-cost, easy-to-use, sensitive, and portable reader, and disposable cartridges for numerous analytes. Such products are intended for both the point-of-care (POC) and OTC markets.

Technology Platform

The IVD industry has recognized the lateral-flow immunoassay as a good technology platform for the POC and OTC markets because of its low-cost and user-friendliness. Most commercial IVD products using an immunoassay platform provide qualitative and semiquantitative results. While IVD companies are or have been developing several emerging lateral-flow assay technologies that provide quantitative measurements, none of the technologies are perfect in terms of detection performance and cost. Kimberly-Clark has been developing a detection technology platform that integrates highly sensitive time-resolved luminescence (including fluorescence and phosphorescence) detection techniques into low-cost, user-friendly, lateral-flow assay platforms.

Figure 1. (click to enlarge) A schematic representation of time-resolved luminescence measurement.

Time-resolved luminescence detection techniques have higher detection sensitivity than conventional luminescence techniques (e.g., fluorescence, phosphorescence) due to higher signal-to-noise ratios.1 Compared with standard luminescence detection methods that separate the luminescence of interest from the background signal through wavelength differences, time-resolved luminescence techniques separate the luminescence of interest from the background signal through lifetime differences. Time-resolved luminescence techniques operate by exciting a luminescent label of a long luminescence lifetime with a short pulse of light, and waiting a brief period of time (e.g., 10 µs) for the background and other unwanted light to decay to a low level before collecting the remaining long-lived luminescence signal (see Figure 1).

The excitation, delay, and collecting processes can be cycled and summed to improve further the signal-to-noise ratio. Doing so allows the short-lived signals, including the scattered light and autofluorescence of a sample matrix, to be rejected before the long-lived luminescence from the label can be measured. The ability to eliminate the background is important for luminescence measurements on a lateral-flow test strip, since its membrane-based structure tends to scatter highly the excitation light. Furthermore, the nitrocellulose membranes commonly used in lateral-flow assays have intrinsic fluorescence that also needs to be eliminated. Overall, time-resolved luminescence methods increase the detection sensitivity by two or more orders of magnitude greater than conventional luminescence detection techniques in liquid samples.1

Kimberly-Clark has developed a new class of luminescent nanoparticles suitable for time-resolved luminescent lateral-flow assays.2,3 The nanoparticles have been used to develop disposable lateral-flow assay strips that can be measured by a low-cost, portable time-resolved luminescence reader.4,5 The reader does not use any expensive optical components. In addition, the technology should have a number of different applications, including clinical diagnostics, detection of chemical and biological warfare agents, and food and environmental monitoring.

Probes

One key element in developing highly sensitive and specific lateral-flow assays is to use suitable probes with good physical, chemical, and spectroscopic properties. The probe’s properties dictate many aspects of the reader development, including cost, size, complexity, and performance. For time-resolved luminescent lateral-flow assays, the particle probes should have strong luminescence with a long lifetime (e.g., more than 40 µs) and a large Stokes shift under ambient conditions. They should be able to be excited by commercially available low-cost light sources such as light-emitting diodes (LEDs). The probes should also be monodispersed with correct sizes and have surface functional groups for surface covalent tagging of recognition molecules such as antibodies. In addition, they should be chemically, physically, and photochemically stable.

Europium chelate-based particles are an obvious probe choice for an initial investigation. Europium particles have strong absorption at 365 nm, strong fluorescence at 615 nm, a long lifetime of 500 µs, and a huge Stokes shift of 245 nm. Monodispersed europium particles with surface functional groups such as carboxylic acid are commercially available from several companies such as Molecular Probes. However, two major properties have made them less than ideal for time-resolved luminescent lateral-flow assays. On one hand, the particles can be effectively excited only by a 365-nm UV light source, which is expensive. On the other hand, the particles are subject to severe photobleaching under UV irradiation. In addition, due to the UV excitation at 365-375 nm, they may not be suitable for consumer-based IVD products for safety reasons. Nevertheless, they are still useful for time-resolved luminescent lateral-flow assays.

Kimberly-Clark has attached several antibodies covalently to europium particles to make antibody conjugates. The conjugates were captured by other antibodies immobilized on the detection zone of a lateral-flow assay strip through an analyte in a sample. When 365-375 nm of UV light from either a fluorometer or a UV LED excites the europium particles captured on the detection zone, strong scattered light from the excitation light and fluorescence of the nitrocellulose membrane extends 400-650 nm. Such strong scattered light makes it difficult to separate the relatively weak signal of the europium particles at 615 nm through wavelength-based separation techniques (e.g., optical filter).

However, the fluorescence signal of the europium particles at 615 nm can be separated from the scattered light and fluorescence of the nitrocellulose membranes through time-resolved techniques. For example, all the background signals such as the scattered light and fluorescence of the nitrocellulose membranes decay to almost zero after a 40-µs delay, while the fluorescence signal of the europium particles remains strong after the time delay because of its long lifetime (500 µs). Kimberly-Clark’s experiments have also demonstrated that the autofluorescence of whole blood and serum can also be rejected by the time-resolved luminescence measurements. Such a complete elimination of background cannot be achieved through conventional fluorescence measurements in which wavelength differences separate the luminescence of interest from the background because of the wavelength overlap.

Although europium particles have many attractive properties as probes for time-resolved luminescence lateral-flow assays, the particles can be excited only by a 365-nm UV light source. As mentioned previously, an intense light source at 365 nm is not inexpensive, and the UV light is not safe for consumer-based IVD products. Furthermore, the 365-375-nm light has limited transmission through nitrocellulose membranes. Such shortcomings limit the detection sensitivity of using europium particles as probes for time-resolved luminescence lateral-flow assays.

Figure 2. (click to enlarge) Kimberly-Clark proprietary phosphorescent probes (Pt-based phosphorescent particles).

Because of the lack of suitable commercial probes, Kimberly-Clark developed a new class of monodispersed and surface functional phosphorescent nanoparticles with ideal physical and spectral properties for time-resolved luminescent lateral-flow assays. The Pt-based particles exhibit strong phosphorescence at 650 nm with a 100-µs lifetime (ex at 390-410 nm), and the Pd-based particles have strong phosphorescence at 670 nm with a 500-µs lifetime (ex at 400-420 nm) under ambient conditions (see Figure 2). These particles also have a large Stokes shift (280 nm for both particles).

The advantages of these particles compared with europium particles include minimal photobleaching, and the ability to apply cheap and powerful light sources such as LED for effective excitation. Furthermore, the background fluorescence is lower for both common biological matrices and nitrocellulose membranes when excited at 390-420 nm, rather than 365 nm. Although transmitting 390-420 nm of light through nitrocellulose membranes is also not ideal, it is better than 365 nm of light, making it more suitable for transmissional mode measurements. The particles have covalently tagged a CRP monoclonal antibody and provided a sensitive time-resolved lateral-flow assay for CRP.

Disposable Lateral-Flow Strips

Figure 3. A disposable lateral-flow strip in a sample holder with three channels.

The antibody conjugates of the phosphorescent nanoparticles were made using standard covalent attachment chemistry. The nitrocellulose membrane-based disposable strips were prepared using standard techniques. The strips consist of a sample zone, a detection zone with an immobilized antibody, a calibration zone, and a wicking zone. In contrast to the opaque supporting plastic materials commonly used for nitrocellulose membrane-based lateral-flow strips, a custom-made supporting material was used for Kimberly-Clark’s strips. The supporting material has high transmittance for visible light (e.g., more than 500 nm) and low transmittance for UV light (e.g., less than 450 nm). In essence, the backing material acts as both a supporting material and a cheap cutoff optical filter. The strip is put into a sample holder before sample application (see Figure 3). After the sample is applied to the sample zone, the strip is inserted into the cartridge holder for measurement and data processing.

Detection Mode

Vigorous testing and evaluation has found that transmission time-resolved luminescence measurement on nitrocellulose membrane-based lateral-flow strips offers more advantages compared with the more common reflectance mode luminescence measurement.6 Kimberly-Clark’s first-generation reader was constructed by applying the transmission measurement mode to demonstrate the feasibility of low-cost time-resolved luminescent lateral-flow assays. Although this mode results in some loss of luminescence of interest because of scattering in the nitrocellulose membrane, it nevertheless allows improved collection efficiency in a practical IVD device.

Furthermore, the simple layout of the reader’s optical components due to the transmission mode facilitates the implementation of low-cost mass production. For Kimberly-Clark’s implementation, in order to improve the transmission efficiency of the test strip, the nitrocellulose membrane is attached to a supporting backing that is opaque to UV excitation but transparent to luminescent emission. By selecting one of several inexpensive, commercially available backing materials as a blocking filter that is integral to disposable test strips, the need for external filters is eliminated.

Figure 4. (click to enlarge) A layout for the optical heads.

Because the phosphorescent particles have a strong absorption spectra (390-420 nm), the reader used 390-nm LEDs as excitation light sources. Standard silicon photodiodes that are insensitive below 400 nm were used for detection. The optical head design involved three channels: the detection line, membrane background, and calibration line on a lateral-flow test strip (see Figure 4). Each channel was equipped with an LED on the membrane side for excitation and two photodiode detectors. The LED is driven by a programmable current source that can be pulsed with a rise or fall time of 10 ns.

The excitation waveform has a rectangular shape with a duty cycle of 10% and a repetition frequency of 2 kHz. A photodiode on the backing side measures the signal luminescence and is connected to a high gain transimpedance amplifier (105 V/A) that is designed to have a bandwidth exceeding 10 MHz. A second photodiode on the excitation side measures nonsignal emission from the nitrocellulose membrane on the reflection side in order to compensate for variations in the LED output power. It is connected to a low gain transimpedance amplifier (103 V/A).

The reader prototype consists of an analog circuit board connected to an optical head that contains the LEDs, detectors, and a receiver slot for the test strips. A computer-interfaced signal generator and data acquisition board are connected to the analog circuit board via breakout connectors.

In addition, the reader prototype consists of three key parts: the optical heads, signal conditioning electronics, and data acquisition equipment. The optical head includes a test strip mount, photodiode holder, LED holder, and custom optical components designed to maximize production and collection of fluorescence.

The signal conditioning electronics includes the LED drivers, a photodiode output conditioning circuitry, and an interface to the data acquisition equipment (computer). It also contains the power supply and/or battery that will power the unit. The electronics control up to six photodiodes and three LEDs that are organized into three groups to monitor three specific areas of a fluorescent test strip: the control line area, test line area, and membrane background. Each group consists of two photodiodes and an LED. One photodiode collects luminescence from the targeted area, and the other monitors the brightness of the LED in the group.

The data acquisition equipment includes an oscilloscope and a signal generator. The signal generator outputs waveforms to the LED drivers in the signal conditioning electronics to control the LEDs. The oscilloscope captures outputs from the signal conditioning electronics and transfers the captured data to a computer for further processing. The unit has been assembled with a working analog-to-digital convertor for measurements. Figure 5 shows the reader (a computer is not shown).

Reader Performance

Figure 6. (click to enlarge) A typical time-resolved luminescence profile from the reader.

The quality of the excitation square pulses from the prototype reader with 390 nm LEDs was very good. The square pulses decay rapidly to background in less than 10 µs, which is less than the lifetime of the phosphorescent particles. Figure 6 shows the time-dependent signal profile of one pulse for a number of lateral-flow strips captured with different amounts of Pt-based phosphorescent nanoparticles. The signal profiles were corrected by the background. More than three orders of magnitude of linear response were observed for the phosphorescent nanoparticles on nitrocellulose membrane-based strips.

Figure 7. (click to enlarge) A linear response of phosphorescent nanoparticles on lateral-flow strips.

The reader also has good detection sensitivity for the phosphorescent nanoparticles on lateral-flow strips. Figure 7 shows the luminescence signal at 40 µs delay time versus the amount of Pt-based particles. The reader’s detection sensitivity was estimated to be 2.5 ng of particle per device. Higher detection sensitivity should be achieved by summing the signals from a number of pulses and integrating all the signals from a period of delay time (e.g., 10-100 µs delay) rather than a signal at a certain delay time.

Applications

Figure 8. (click to enlarge) A dose response of CRP.

CRP has demonstrated the application of the time-resolved luminescent lateral-flow assay for analyte quantitation. Figure 8 shows the time-resolved luminescence signal at 40 µs delay time as a function of CRP concentrations on lateral-flow assay strips. The detection sensitivity for CRP can reach 20 picograms per device in diluted blood. The detection sensitivity is limited by the binding strength of the antibodies rather than the detection method. Furthermore, the lateral-flow devices for CRP detection are far from optimized. Higher detection sensitivity will be feasible if antibodies of higher binding strengths are available and the conditions are optimized.

Reader Cost

Time-resolved luminescence techniques allow complete elimination of expensive band path optical filters for the reader. Furthermore, the unique transmission measurement mode makes costly mirrors and optical lenses unnecessary. The cost of producing the reader is therefore much cheaper than a reader for conventional luminescence (e.g., fluorescence). Kimberly-Clark’s current first-generation reader prototype was built as a research tool, which allowed tailoring of many parameters for the lateral-flow strips. The reader is connected to a computer to allow adjustments of many different measurement parameters and data processing. The prototype reader can be converted into a self-contained and portable unit that can process and display the data on its own screen. For such a reader, the cost is estimated to be less than $15 for the bill of materials for all the components for a volume of more than 100,000 units.

Conclusion

This article summarized the design, development, and evaluation of a time-resolved luminescent lateral-flow assay technology. Europium chelate-based particles have significant limitations as probes for applications on time-resolved luminescent lateral-flow assays. Because of the lack of suitable probes, Kimberly-Clark has developed proprietary phosphorescent nanoparticles that are suitable for time-resolved luminescence measurement on lateral-flow strips. The company has also developed a low-cost and compact reader that can measures the time-resolved luminescence signals from designated zones of a lateral-flow strip. The reader is inexpensive and does not use any expensive optical components such as band pass filters, lenses, and mirrors. The time-resolved luminescent lateral-flow assay technology has been demonstrated to be capable of providing sensitive analyte detection with a large linear dynamic range. The detection platform should find a wide range of potential diagnostic applications.