POINT-OF-CARE TECHNOLOGIES
Part 1: Devices and applications for lateral-flow immunodiagnostics
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Figure 1. (click to enlarge) A 90-gram optoelectronic unit (a) and detail of the engineered
interior (b) is the core of the handheld and mobile systems by ESE Embedded System Engineering GmbH (Stockach, Germany). The unit includes two excitation sources and two detectors for different wavelengths. |
Part one of this article describes portable devices that demonstrate precision when combined with lateral-flow test strips using clinical sample media (e.g., urine) in applications such as MDMA drug testing. Such testing platforms provide full documentation of quantitative data and avoid subjective interpretation of test results. This article also discusses approaches to miniaturizing modular devices, which allows rapid market access. Part two of this article will focus on handheld nucleic acid–based testing platforms and show examples of antibiotics resistance testing.
Demand for Handheld Testing Platforms
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Table I. (click to enlarge) Some drivers, rationale, and concluding features for handheld and mobile test platforms.
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More than a thousand infectious diseases currently known to modern medicine require diagnostic testing.1 There is also a need for testing for drug consumption, toxins in food and agricultural products, genetically modified organisms, cancer markers, biothreat agents, allergic and immune response parameters, and human identification. Because nonprofessional laboratory personnel perform many of these tests either in the field with handheld devices or in a lab on benchtop instruments, such platforms should be simple and easy to use. Some important factors that also drive the development and use of such systems include convenience, decreased risks, privacy, epidemiological resolution, lack of power availability, and space limitations (see Table I).
Other key factors in developing POC test systems are turnaround time and operational cost savings, which can be increased significantly if sample storage and transportation can be avoided. Mobile and handheld devices allow targets to be probed in the field, which overcomes time-consuming sample transportation and storage requirements. For example, an outbreak of a contagious disease in a military camp can be controlled if monitoring of the epidemic can be provided through on-site testing devices. Bedside testing allows test results to be delivered in an emergency room, ambulance, or hospital, and prevents sending samples to a reference laboratory.
The demand for mobile testing devices comes from various segments in the POC market such as physician offices, government security agencies for biothreat agent detection and epidemiological monitoring, hospitals for bedside testing, and home-care providers for telemedicine. Devices serving these markets need to be accurate, robust, affordable, easy to operate, and provide rapid turnaround time (see Table I).
Simplifying Sample Preparation and System Operation
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Table II . (click to enlarge) Steps and time involved in sample testing.
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Extensive and time-consuming sample preparation is one of the primary limiting factors in adapting diagnostic tests to home care and POC settings. When developing POC systems, the entire work flow for testing needs to be considered, not only the actual assay, including steps such as sample storage and transportation, which can take days to weeks in some cases (see Table II). Extended and inaccurate sample storage and transportation may lead to degraded and contaminated samples. This could result in elevated rates of false-positive and false-negative test results, and increased risk of infection for lab personnel due to the number of sample-handling steps. It also places cost burdens on the healthcare system.
Mobile systems must meet all of the requirements listed in Table II. But even though mobile tests avoid sample storage and transportation altogether, mobility alone still does not address the sample preparation part. While many groups have tried to miniaturize the sample preparation steps in current test procedures, ESE Embedded System Engineering GmbH (Stockach, Germany) combines its mobile and handheld devices with assays, which avoids the most time-consuming steps such as extensive sample preparation. This allows for on-site testing and rapid time to result, leading to decreased risk, both for personnel and of generating false-positive or false-negative results.
Mobile systems must ideally be able to take a sample directly and be simple to operate. There are only a handful of test procedures that may fulfill this paradigm. For example, lateral-flow assays have been shown to work directly from blood, urine, and saliva.2 They also meet all of the other criteria for field-based tests (see Tables I and II). Another method is a nucleic acid amplification technique called recombinase polymerase amplification (RPA), which will be discussed in part two of this article.3
Device Design and Miniaturization
Miniature Optoelectronic Bench. ESE has developed and manufactures handheld and mobile devices for automated testing that meet the parameters listed in Tables I and II. The core of the devices’ sensors is a 90-gram miniature optoelectronic unit (see Figure 1). This unit is configured with two excitation sources (365 and 660 nm) and two detectors suitable to measure two emission wavelengths (460 and 720 nm).
The optimized miniature microoptical bench is the core technology of such devices as the handheld lateral-flow scanners (FluoScan and GoldScan), and the mobile FluoSens devices. The FluoSens detectors are fluorescence sensors that are used for fluorescence detection of surfaces or liquids in a single tube, cuvette, or microtiter plate.
Rapid Prototyping Tools. The miniature devices discussed in this article are too small to be evaluated by a breadboard construction on an optical bench. Instead, optical simulation tools such as Zemax by Zemax Development Corp. (Bellevue, WA) are used to virtually build a model, optimize it on screen, and subject it to rapid prototyping. This approach allows fast turnaround in days.
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Figure 2. (click to enlarge) Simulations of miniature optical benches
using Zemax by Zemax Development Corp. (San Diego). Shown in colors are the pathways of light emitted by a sample (a and b) or a light source (c). |
The insertion of filters and light sources can then easily be done on a customized basis. Figure 2 shows the pathway of light emitted by a sample passing various filters and being projected onto the detector photodiode.
Modular Design. Due to the modular design of ESE’s lateral-flow reader, the device can be configured for fluorescence readout in the
FluoScan or gold-bead-based colorimetric readout in the GoldScan, and can be adjusted to different lateral-flow cassette formats. The lateral-flow cassette carriage can be substituted by a tube rack that is suitable to scan fluorescence kinetics in multiple tubes, such as for real-time nucleic acid amplification, PCR endpoint readout, and DNA, RNA, and protein quantification using fluorescent reagents (see Figure 3).
Such a modular design approach allows for cost-efficient and rapid customization, as well as for exploiting the device for multiple applications. Both flexible mobile systems in a suitcase as well as handheld and battery-operated systems have been built (see Figure 3). Wireless data transfer can also be implemented upon request. The devices are constructed for robustness and are easily operated by a single button.
The device’s internal memory and display provide data output on-site; up to 2000 scans can be saved without a computer. Such data output can be qualitative, semiquantitative, quantitative, or action-based.
Lateral-Flow Test Applications
Lateral-flow tests are very affordable because they can be read by visually inspecting a strip without a device. However, there has been a drive toward automated lateral-flow test readouts due to the current limitations of gold-bead-based colorimetric immunochromatographic techniques. Such limitations include the following: subjective interpretation of results, nonquantitative results, lack of automated electronic documentation, and sensitivity limits.
The lack of lateral-flow test readers in the past is surprising and has limited the exploitation of lateral-flow techniques for particular targets and applications. A recent trend in the IVD industry is generation of quantitative and highly sensitive test results. By doing so, the goal is to automatically document data and avoid subjective interpretation of results, which decreases the number of false positives and false negatives. This has been previously approached with magnetic-bead-based lateral-flow assays, which required sophisticated instrumentation.4
More recently, a variety of handheld readers for immunochromatographic test strips were developed, most for reading colorimetric strips. Fluorescence-based lateral-flow tests have been shown to be more sensitive for particular applications than gold-bead-based colorimetric tests, which allows the potential for exploiting this technique for new applications and markets.5
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Figure 4. (click to enlarge) Principle of fluorescence lateral-flow test
readouts. The sample is applied to the strip, the strip
put into the reader, and the appearing control and test
bands are scanned.
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The operation and measurement principle of a handheld lateral-flow scanner is simple (see Figure 4). A sample is applied to a sample filling port and flows through the strip. After a few minutes, fluorescence or gold-bead-based colorimetric control and test bands appear. The absence or presence of the bands indicates a positive, negative, or invalid test result. The strip is placed in a reader and moved over a light source. When the bands are illuminated by the light, a fluorescence or reflection signal is recorded in the reader through the same optics because of the confocal design. The quantitative data are recorded, and one scan takes 1 second. Data for up to 2000 scans are stored internally in the device, which allows the battery-operated device to be a stand-alone product. Connectivity to a computer is provided by a USB port or a wireless data transfer.
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Figure 5. (click to enlarge) Lateral-flow MDMA test using fluorescent
latex beads. |
In one experiment, MDMA testing was done using fluorescent latex beads. The test was performed using a lateral-flow test strip and the FluoScan device. Samples of MDMA with concentrations of 0, 125, 250, 500, 750, and 1000 ng/ml MDMA were spiked into a UriSub urine substitute by CST Technologies (Great Neck, NY) and applied to a test strip. After 5 minutes, the test strip was scanned using the FluoScan device. The test result showed a linear regression with a correlation coefficient of R2 = 0.9958. This demonstrates quantitative, sensitive, and accurate test results directly from urine without prior sample preparation steps (see Figure 5).
In order to check the device’s reproducibility without varying the biochemical test involved, a reproducibility study was performed in another experiment. A single lateral-flow strip cassette was scanned 20 times. The standard deviation was determined at 0.039% for the test band and 0.040% for the control band. A similar experiment was also performed, and peak positions rather than peak intensities were recorded. Standard deviations were determined at 0.40% and 0.30% for the test band and the control band, respectively. This demonstrates precision and reproducibility of the device.
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Figure 6. (click to enlarge) Lateral-flow test strip scanned using the
FluoScan device. The test and control bands are visible.
Two samples are positive and one is negative, as can
be seen from the missing test band.
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Figure 6 shows examples of data scans of lateral-flow test strips that were scanned using the FluoScan device. A negative sample and low-positive and high-positive analyte concentrations were tested.
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Figure 7. (click to enlarge) The software and parameters are shown to record and interpret
the data when using the FluoScan lateral-flow reader. The calculations include
background correction, determination of peak intensities of test and control
band, and cutoff levels for peak intensities to determine a positive, negative,
and invalid result.
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The fully automated software calculates and displays the sample results. It can list the data qualitatively, semiquantitatively, quantitatively, or action-based. Figure 7 shows the software and parameters used to record and interpret the data automatically. Raw and interpreted data are also generated in an Excel format (see Figure 8). The devices can be used without a computer since they are battery-operated, and data are displayed on the internal display and saved in the internal memory.
Quantitative results can be obtained by using either an internal standard or an external standard. In lateral-flow assays, the control band can be used as an internal standard. In this case, the ratio of peak intensities of the test band over the control band can be used as a quantitative measure as this ratio is a constant at a defined analyte concentration.
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Figure 8. (click to enlarge) A data table is generated in Excel format for both qualitative and
quantitative results. The quantitative results shown here are given as peak
intensity of test band relative to control band (in %, highlighted in green).
Qualitative data are highlighted in gray. An external standard curve can also
be used to conclude in quantitative results.
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This type of ratio analysis has several advantages. First, both the standard (control band) and the analyte (test band) are scanned under the same conditions at the same time during the same scan. For example, changes in parameters such as excitation efficiency or light spot homogeneity apply to both the test band and the analyte band.
Second, variations and shifts in baseline intensity due to the biological matrix applied to the lateral-flow test strip are eliminated as this effect is cancelled out when calculating the ratios of intensities. For some applications, only the ratio analysis leads to a quantitative result. The second method using a calibration or standard curve (external standard) yields reproducible quantitative results if there are negligible shifts in baseline intensity. Of course, integrals rather than peak intensities could also be used for data interpretation.
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Figure 9. Design study of the
next generation mobile FluoScan serial device. |
Transitioning the current scanners into more-affordable devices designed for mass production is close to being finalized. Figure 9 shows a rendered design of the next generation of mobile devices. The device has two main parts. The front part in white contains all the components to operate and control the device. This part is generic and can be produced in high volumes. The rear part in grey contains the carriage for the lateral-flow cassette. This part is interchangeable and can be substituted with other carriages suitable to hold tubes, microfluidic chips, cuvettes, gels, microscope slides, microarrays, or other sample formats. An x–y stage can scan in two dimensions. It also contains the sensor, which can be easily adapted to the requested wavelengths. This modular design allows rapid market access for various applications and sample formats.
Other applications for handheld lateral-flow scanners that are not discussed in this article include the entire range of immunodiagnostics such as on-site testing of infectious diseases, antibiotic resistance, hormone titre, immune response, drugs, food, agricultural, and routine laboratory measurements, just to name a few examples. As the sensors of lateral-flow devices can easily be switched between fluorescence and reflectometric measurement modes, colorimetric tests such as ELISA procedures can also be done. The systems described in this article are suited to serve both the POC and home-care markets due to their specificity, speed, ease of use, low weight, and small size.
Conclusion
Battery-operated, handheld, and mobile diagnostic testing platforms have been built and can provide sensitive, accurate, and specific results as well as rapid turnaround time, operational and physical robustness, and affordability. The assays work directly from clinical samples such as urine and blood. Due to their mobility, the platforms avoid sample storage and transportation. And due to their ability to work directly from clinical samples, they avoid most sample-preparation processes, which are the most time-consuming and troublesome steps in every diagnostics procedure. Having covered examples of lateral-flow immunodiagnostics in this part of this the article, the second part will cover handheld nucleic acid–based testing platforms.
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Konrad Faulstich, PhD,
MBA, is head of business
development at ESE
Embedded System
Engineering GmbH
(Stockach, Germany). He can be reached at konrad.faulstich@ese-gmbh.de.
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Michael Eberhard is chief
electronics engineer at
ESE Embedded System
Engineering GmbH. He can be reached at
michael.eberhard@ese-gmbh.de. |
Roman Gruler is head of
R&D at ESE Embedded
System Engineering
GmbH. He can be reached at roman.gruler@ese-gmbh.de.
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Klaus Haberstroh is CEO
and cofounder of ESE
Embedded System Engineering
GmbH. He can be reached at klaus.haberstroh@ese-gmbh.de.
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References
1. WB Karesh and RA Cook, “The Human Animal Link,” Foreign Affairs 84, no. 4 (July/August 2005).
2. B O’Farrell and J Bauer, “Developing Highly Sensitive, More-Reproducible Lateral-Flow Assays, Part 2: New Challenges with New Approaches,” IVD Technology 12, no. 6 (2006): 67–75.
3. O Piepenburg et al., “DNA Detection Using Recombination Proteins,” PLoS Biology 4 (2006): 1–7.
4. RT LaBorde and B O’Farrell, “Paramagnetic-Particle Detection in Lateral-Flow Assays,” IVD Technology 8, no. 3 (2002): 36–41.
5. J Bonenberger and M Doumanas, “Overcoming Sensitivity Limitations of Lateral-Flow Immunoassays with a Novel Labeling Technique,” IVD Technology 12, no. 4 (2006): 41–46.
