1. Introduction
Point-of-care diagnostics (POCD) for health monitoring involves the evaluation of indices from human health through tests performed outside of the clinical laboratory, typically right at the site of patient care [
1,
2,
3,
4]. While the advantages of POCD such as rapid diagnosis, operational efficiency, and costs have been well established [
2], widespread implementation of this technology has not yet been achieved [
5]. One of the main limitations of POCD is the requirement of unprocessed test samples drawn from patients to provide the desired sensitivity and specificity for conducting reliable assays in situ. However, most of the currently validated clinical assays are carried out using plasma, prepared from whole blood samples inside sufficiently equipped laboratory conditions. Here, we develop a biosensor platform that could be reliably used for instantaneous detection of target molecules from non-invasively obtained bodily fluid samples for real-time point-of-care testing applications.
Although whole blood assays are generally considered as the gold standard for bioanalysis, such an approach may not be convenient to implement under all circumstances. In general, high-risk patients under intensive care or long-term hospitalization suffering from extremely sick conditions may not always be able to bleed enough quantity of blood. Possible nerve damage during venipuncture [
6] could render the procedure extremely painful, traumatic and psychologically disturbing for the patients, such that even skilled phlebotomists could face limitations to extract enough quantity of blood under such circumstances.
One strategy to overcome the trauma associated with such painful venous blood draw procedures could be to adopt a technique that is less invasive and utilizes smaller volumes, possibly through a finger prick as implemented with some of the commercially available handheld blood glucose monitors. However, a limitation with this approach is that reduced sample volumes call for greater sensitivity of the measurement setup and may also not always contain the necessary distribution of the target molecules under all circumstances. This could lead to inconsistent readings and false negative results [
7]. Furthermore, common clinical problems with whole blood assays such as hemolysis or coagulation could also result in an inaccurate representation of the analyte concentration [
8].
An alternate strategy would be to eliminate the necessity for whole blood, and use other test samples for carrying out the required bioanalysis. Among the bodily fluids generally used in clinical assays (shown in
Figure 1), those commonly available through non-invasive extraction procedures include urine, saliva [
9] and sweat [
10]. While inducing sweat out of hospitalized patients is practically difficult, urinary extraction may also necessitate additional diuretics which could directly interfere with the measurands, aside from inducing other significant side effects [
11]. Considering these factors, saliva presents a viable alternate to the traditional biofluids used in biochemical analyses.
Saliva is considered as a filtrate of the blood representing the physiological state of the body. Given the ability of molecules from blood to diffuse into saliva, salivary diagnostics is increasingly being recognized as an equivalent to serum analysis [
12]. Research studies using saliva as a bio-diagnostic fluid includes works such as evaluation towards dialysis needs in renal failure patients [
9] and on-site analysis for biochemical factors [
13]. The incorporation of microfluidic methods in bioassays of saliva has further reduced sample and reagent consumption, and has decreased the overall assay times [
13]. Research studies in salivary diagnostics involving microfluidics include measurement of C-reactive protein using an fluorometric immunoassay [
14], on-chip polymerase chain reaction (PCR) system for rapid fluorometric detection of genetic deletion [
15], paper-based device for quantification of the nitrate concentration [
16], detection of thiocyanate through surface-enhanced Raman scattering (SERS) [
17], and spectrometric absorbance detection of NH
3 and CO
2 in saliva as a biomarker for stomach cancer [
18]. In addition, most of the reported microfluidic systems for salivary diagnostics included integrated optical sensing for their bio-detection scheme [
19].
In this work, we present the development of a simple, hybrid integrated optical microfluidic biosensor for rapid analysis of saliva, and we have demonstrated the application of the proposed biosensor format by detecting the presence of potassium from whole, untreated salivary samples. Inspired by the age-old concept of colorimetry published as early as the first half of the 20th century [
20,
21], we hereby show that potassium can be detected from human saliva samples through the principle of optical absorption. A commercially available potassium detection reagent mixed with saliva samples creates turbidity based on the potassium concentration, which could then be measured through the absorption of light passing through this turbid path. We have implemented this optical sensing principle on a lab-on-a-chip platform by integrating colorimetric detection within a microfluidic system that facilitates transportation and handling of the analytes and the reagents. Homogeneous mixing of fluids inside the microfluidic system has been achieved through the integration of piezo-actuated acoustic micromixing. The results of this work show that the proposed hybrid integrated device can be applied to real-time optical biosensing using whole, unprocessed samples, which could be extremely useful for automated point-of-care testing applications.
2. Materials and Methods
2.1. Development of the Integrated Optical Biosensor
This section provides the details of the design, fabrication, integration and packaging of our hybrid integrated optical microfluidic setup. The schematic illustration of the processes is shown in
Figure 2.
2.1.1. Device Design and Fabrication
The biosensor (schematically shown in
Figure 2A) consists of a microfluidic system with two inlets which accommodates two different types of fluids. The fluids are transported to a piezo-actuated (acoustofluidic) micromixer unit, and upon mixing are subsequently transported to the optical detection unit. The configuration of the bulk acoustofluidic micromixer unit was adopted from [
22,
23]. The optical detection unit consists of SU8 waveguides core integrated onto poly dimethylsiloxane (PDMS) (cladding) through the inlet and outlet waveguide channels (as indicated in
Figure 2A). Convex curvatures at the end of the optical channel enable convergence of light into the fluidic channel. Two different types of optical waveguide assembly were designed. In the configuration used in the present work, the output waveguide was fabricated co-axially with the input waveguide. This configuration allowed direct coupling of the input light onto the output waveguide, as required in optical absorption measurements.
To fabricate the devices on PDMS (fabrication and integration processes schematically shown in
Figure 2B), silicon master molds with patterned SU8 (100 μm thickness) were fabricated using standard soft lithography process. Poly dimethylsiloxane (Sylgard 184, Dow Corning, Midland, MI, USA) with prepolymer to curing agent volumetric ratio of 10:1 was mixed, degassed and poured onto the silicon mold. The setup was left to cure at 70 °C for four hours, after which PDMS was peeled off the mold and diced to create the individual devices. To create optical waveguides, the PDMS devices were exposed to oxygen plasma (2 min, 200 mTorr pressure, 20 cubic centimeter (ccm) flow rate, and 60 W Radio Frequency (RF) power), and thereafter SU8-5 (Microchem, Westborough, MA, USA) with viscosity 290 cSt, and refractive index ~1.6 was allowed to fill in the optical channels through capillary flow. The photoresist was cured by flood exposure to ultraviolet (UV) light (12 mW/cm
2) for 60 s, and the devices were diced thereafter to enable fiber attachment with the SU8 waveguides.
2.1.2. Integration and Packaging
Coupling of the input and the output fibers were carried out under a stereo microscope. FC connectorized tapered lens-ended fiber (OZ Optics, Ottawa, ON, Canada) which gives a spot size of 5 μm, was connected to a broadband laser light source (Ocean Optics, Largo, FL, USA) for optical input. The output collector fiber was SMA end connectorized and coupled to a handheld spectrometer (USB 2000, Ocean Optics, Largo, FL, USA). Fiber strippers and precision cleavers (Newport, RI, USA) were used to remove the buffer layer and cladding around the fiber. The input and the output fibers were positioned in separate five axis fiber positioners on supporting V-grooves, so that each of the fibers and the waveguides can be maneuvered and coupled appropriately for acquiring the maximum optical signal. Maximum optical light coupling into the fiber was ensured by fine-tuning the position of the fiber and by observing the maximum signal from the output fiber coupled into the spectrometer. Thereafter, UV index matching gel (NOA60, Norland International Inc., Lincoln, NE, USA) was applied at the tip of the fiber and the setup was exposed to UV for 60 s to bond the fiber with the SU8 waveguides.
The device was then treated with atmospheric plasma using a handheld plasma cleaner (Plasmaetch, Carson City, NV, USA) and the channels were sealed using a 100 μm thick glass coverslip. Piezoceramic discs (T216-A4NO-173X, Piezo Systems Inc., Cambridge, MA, USA) used for generating the acoustic waves were attached to this glass diaphragm using silver conductive epoxy (also used as the bottom electrode). The top electrode was soldered to the piezoceramic. For ease of handling, the device was attached with a polycarbonate support using double-sided adhesive tape, without damaging the optical fibers. A through hole drilled on the polycarbonate prior to the attachment of the optical-microfluidic chip accomomodates the piezo actuator inside the cavity.
2.2. Chemicals and Reagents
Integrated optical-microfluidic characterization experiments were carried out with de-ionized (DI) water and ethanol (Sigma Aldrich, St. Louis, MO, USA). Prior to introducing fluid samples into the system, the microfluidic channels were flushed with ethanol in order to remove any contaminants or air bubbles. Initial micromixing characterization experiments were conducted by staining the working fluids (DI water or ethanol) with standard food coloring dyes. Preliminary experiments for the characterization of optical absorption with integrated micromixing were conducted using glucose enriched RPMI 1640 cell culture media (A1049101, Thermofisher, Waltham, MA, USA) as the working fluid. Herein, the glucose present in cell culture media was tested to produce an expected colorimetric reaction with Benedict’s reagent prepared according to the protocol described by Cochran et al. [
24]. For colorimetric salivary analysis experiments, potassium colorimetric assay kit (E-BC-K279) was procured from Cedarlane Labs (Burlington, NC, USA). Whole samples of unstimulated saliva were collected from volunteers in centrifuge tubes by a simple spitting method, and the specimens were vortexed thoroughly. Prior to the experiments, protein precipitant and chromogenic agents were prepared as described in the protocol prescribed by the manufacturer. 20 µL of the saliva samples were mixed with 180 µL of the protein precipitant and centrifuged at 1100 g for 10 min. Thereafter, the supernatant was used in the experiments, mixed with a volumetrically consistent chromogenic agent for the colorimetric optical absorbance measurements.
2.3. Integrated Testing and Measurement
The piezoelectric system was driven by an external signal generator (33120A, Hewlett Packard, Palo Alto, CA, USA) and an amplifier (PCB Piezotronics, Depew, NY, USA) where a sinusoidal signal from the signal generator was amplified 20-fold by the amplifier. Typically, the operating voltage of 100 Vp-p was used for piezo-actuation. For continuous flow experiments, liquids were injected at the same flow rates inside the microchannel using syringe pump (KDS-210, KD Scientific, Holliston, MA, USA). In all other cases, liquids were manually pipetted into the microfluidic channels.
The mixing efficiency was calculated based on the change in the pixel values of the respective color fluids.
Optical signals were collected using a handheld spectrometer (USB2000, Ocean Optics, USA). The optical absorbance of the specimens measured by the spectrometer is given by the formula
where,
λ—Wavelength of light used
Aλ—Absorbance
Sλ—Intensity of light passing through the sample
Dλ—Dark intensity
Rλ—Intensity of light passing through a reference medium.
The dark intensity was recorded by measuring the optical signal intensity when the light source was turned off. For potassium measurement experiments, the chromogenic agent was used as the reference medium to measure the reference intensity.
2.4. Imaging and Statistical Analysis
Images were recorded using an OFV-A-534-Cax video camera inbuilt with the single point Laser Doppler Vibrometer (LDV, Polytec, Detroit, MI, USA). Scanning electron microscopy (SEM, SU3500, Hitachi Hi-Technologies, Tokyo, Japan) was conducted using standard protocols for variable-pressure imaging mode (3.0 kV, 30 Pa,) allowed SEM observations of the PDMS devices without the need for additional sample manipulation or conductive coating. The images were processed using ImageJ (National Institute of Health, Bethesda, MD, USA), following standard protocols. All statistical comparisons were made using one- or two-way analyses of variance (ANOVA) with Tukey post-hoc comparisons (Prism; GraphPad Software, La Jolla, CA) with p-values < 0.05 considered significant, and graphical data reported as means ± standard error for at least n = 3 experiments.
4. Discussion
Salivary diagnostics is being increasingly touted as a suitable alternative to whole blood assays. However, the study of salivary functions could also be challenging because of the high physiological variability of saliva in comparison with other bodily fluids [
38]. However, given that the saliva samples can be obtained non-invasively, patients are much more likely to cooperate for frequent extraction of saliva, for performing assays regularly. We also observed this trend among the volunteers, who were unanimously relieved with the painless approach of collecting saliva samples for our experiments. Nevertheless, to exploit this convenience of salivary analysis, it is also equally important to develop a suitable automated point-of-care testing methodology that would enable rapid diagnostics to reduce the time and effort normally put into laboratory-based assays. The cumulative results of our present work demonstrates that the hybrid integrated optical biosensing principle proposed herein could be readily applied to potassium measurement from whole saliva samples for real-time point-of-care testing applications based on the principle of colorimetry.
The colorimetric optical absorption-based detection principle presented here is simple and can be easily extended to any other fluid sample. While the results of traditional point-of-care colorimetric assays reported to the naked eye (such as pregnancy tests or blood glucose tests), integrating smart-phones [
39] for automated testings have lately been gaining huge popularity. But, the implementation of the colorimetric technique would not be effective unless uniform, controlled mixing of samples with the appropriate chromogenic agents is achieved. The acoustofluidic actuator thus lends a major advantage to this device for achieving the required homogenous sample mixing. The bulk acoustic wave micromixer configuration described in this work is simple and cost-effective to fabricate. For the device setup presented here, the ease of operation and feasibility of seamless hybrid integration with other complementary modules of the biosensor platform lend advantages to the bulk acoustic wave micromixer over the popularly used surface acoustic wave micromixing formats. However, mixing efficiency could be further improved by adopting simple modifications to the existing setup, such as incorporation of passive microfluidic elements, surface treatment resulting in increased hydrophobicity [
40], and using acoustic index matching glues to improve piezoacoustic energy transfer. Our initial experiments showed that the cavitation-based acoustofluidic micromixing can be used with a wide variety of fluids with different physical properties such as miscibility, viscosities, vapor pressures etc. (
Supplementary Figure S1B). While the present work focused only on cavitation-enabled fluid mixing, it was also observed during the initial characterization experiments that the cavitation phenomenon also induced other physical effects in the fluids. For example, it was possible to take advantage of the energy released from collapse of the cavitation bubbles for cleaning the surface of the microfluidic channels to get rid of non-specifically bound adherent particles [
26]. This functionality could be extremely useful for conducting biological assays repeatedly without cross-contamination inside the microfluidic channels.
However, there are a few shortcomings to the present approach. Firstly, the device format in its present state does not enable the realization of cheap, disposable biosensors. But, since the device is sterilizable for repeated use, integrating light sources and sensor units on-chip along with suitable optoelectronic packaging could enable further help with the miniaturization of this device for developing a more cost-effective version. Secondly, optical absorption-based detection may hit the limits when conducting assays for measuring extremely low analyte concentrations in the samples. Despite the relatively poor sensitivity of microfluidic absorbance detection compared to fluorescence, the principle of working of optical absorption and its instrumentation simplicity gives it an advantage in applications requiring point-of-use analysis. Also, wherever applicable, it is possible to carry out a comprehensive study of time-dependent biochemical interactions using the optical absorption method. One can also take confidence from the fact that a number of absorbance-based microfluidic point-of-care products are commercially available [
41]. Our device design also allows for implementing more than one optical sensing principle on the same platform. In an alternate waveguide arrangement to that used in the present work, the output waveguide was designed at an angle with respect to the inlet waveguide so that light is not directly coupled onto the output waveguide. This configuration, although not used in this work, could be useful for sensitive low absorption measurements or fluorescence-based optical detections where specific emission wavelengths are sensed spectroscopically without being saturated by the excitation wavelengths. Thus, overall our present work clearly demonstrates the feasibility of developing integrated microfluidic optical detection systems for rapid biosensing and real-time diagnostics, which could be incorporated into the next generation point-of-care devices.
5. Conclusions
Saliva is a non-invasive biofluid which is easy to collect, transport, and store. Because of its accessibility and connection to systemic diseases, saliva is one of the best candidates for the advancement of point-of-care medicine, where individuals are able to easily monitor their health status by integrating the salivary diagnostics with suitable biosensing systems. Our present work on the development of a hybrid integrated optical detection based biosensor with integrated micromixing further asserts that colorimetric salivary analysis can be conducted in real time in a dependable, noninvasive, simple, and rapid manner using whole, unprocessed saliva samples with the appropriate chromogenic agents, to screen for any required minerals and metabolite values Thus, this approach could come in handy for real-time point-of-care screening applications for monitoring high-risk hospitalized patients.