An integrated microfluidic chip for non-immunological determination of urinary albumin
This study presents an integrated microfluidic chip for non-immunologically determining the concentrations of albumin in clinical urine samples. This microchip integrates membrane-type micromixers and a fan-shaped micropump capable of simultaneously and precisely delivering assay reagents to react with 6 urine samples in one single operation. The experimental results show that the coefficient of variation in the pumping rate is 2.42%. More importantly, using this unique chip design, only 2 electromagnetic valves are required for the actuation of the micromixer and the micropump. The working range of the proposed microchip is 2–200 mg/L of albumin, which covers the range of interest for the determination of microalbuminuria. Moreover, statistical analysis show that the results obtained by the proposed microchip are in good agreement with the conventional detection method, based on immunological assays. This simple, inexpensive and microchip-based platform presents a promising alternative to conventional immunological assays for measurement of urinary albumin, and is well suited for clinical applications.
KeywordsMicroalbuminuria Immunoassay Microfluidics Micropump
albumin excretion rate
biological field-effect transistor
coefficients of variation
enzyme-linked immunosorbent assay
human serum albumin
limit of detection
metal oxide semiconductor field effect transistor
molecularly imprinted polymer
normally closed valves
quartz crystal microbalance
Human serum albumin (HSA) which is composed of one long polypeptide chain (66.5 kDa) is a major constituent of plasma proteins (Dockal et al. 2000). A kidney is capable of retaining beneficial proteins in blood such as albumin and filtering out waste components. However, when the kidneys are severely damaged, albumin may appear in the urine. The quantitative determination of urinary albumin is of great importance because it can provide critical information on different aspects of renal function and many diseases have been linked to an increased albumin excretion rate (AER) (Waller et al. 1989; Chase et al. 1991). A slightly increased AER is termed as microalbuminuria (MAU) and is recognized as an early predictor for nephropathy in patients who suffer from diabetes and hypertension (Mogensen 1987; Vigstrup and Mogensen 1985). In addition, a number of studies suggest that MAU is also an indicator of cardiovascular diseases in nondiabetic individuals (Lydakis and Lip 1998; Haffner et al. 1990). Since diabetic nephropathy is potentially reversible at the early stage (Viberti et al. 1979), early detection and control of MAU can reduce the risk of nephropathy and renal failure. Thus, routine monitoring of MAU is essential for patients at high-risk for diabetes. For the monitoring of MAU, the range of interest of urinary albumin concentration is between 2–200 mg/L, particularly the 15–40 mg/L range that covers the usual cutoff limits between normal and increased albumin excretion (Rowe et al. 1990).
Since a urine sample is very complex and the urinary albumin concentration is relatively low for MAU determination, several immunoassay-based methods such as a solid-phase fluorescent immunoassay (Chavers et al. 1984), an enzyme-linked immunosorbent assay (ELISA) (Aybay and Karakus 2003), piezoelectric immunosensors (Navrátilová et al. 2001), a liposomal immunoassay (Frost et al. 1996), magnetic immunoassay (Lu et al. 2006), and a molecularly imprinted polymer (MIP)-quartz crystal microbalance (QCM) sensor (Hu et al. 2005) have been developed to determine urinary albumin due to these specificity and sensitivity issues. Although these immunoassay-based methods are reliable for urinary albumin determination, the costs, mainly attributed to antibodies required for immunoreactions, are high and may limit their applications in local laboratories for routine monitoring of MAU.
Recently, microfluidic systems have attracted considerable interest and shown their great potential for chemical analysis and biomedical applications due to unique advantages including fast analysis time, high throughput, low sample/reagent consumption, disposability, portability, reliability, and their capability for integration and automation (Manz et al. 1990; Liao et al. 2005). Recently, several microfluidic devices have been developed to determine urinary albumin concentrations, such as a microchip electrophoresis (Chan and Herold 2006) and a biological field-effect transistor (BioFET) (Park et al. 2008). The assay based on microchip electrophoresis used a commercial assay kit for quantifying urinary albumin by adding chicken albumin as an internal calibrator. There, sodium dodecyl sulfate in the buffer binds to the albumin. Fluorescent dye then binds to the sodium dodecyl sulfate micelles as an indicator of albumin concentration. Although this method quantifies urinary albumin with good sensitivity, precision, and accuracy, an electrophoresis separation step is required. The BioFET is a metal-oxide-semiconductor field effect transistor (MOSFET)-type protein sensor with gold deposited on the gate insulator. The gold on the gate surface was first chemically modified by a self-assembled monolayer (SAM), followed by immobilizing anti-albumin antibody to conjugate with albumin in the samples. The drain current was then modulated by the albumin bound to the anti-albumin and the concentration of urinary albumin was estimated according to the current variation ratio. Although the BioFET provides advantages such as small size, fast response, high reliability, and it is potentially portable, it is an antibody-based sensor and hence its application in routine monitoring of urinary albumin is still limited due to the costs attributed to the antibody reagents.
In order to simplify the procedures and reduce the costs for urinary albumin determination, a non-immunological method based on a dye-binding assay has been developed. The method is based on the characteristics that fluorescence probes, namely albumin blue (AB) 633 and AB 670, are highly selective for HSA and the fluorescence intensity is enhanced by orders of magnitude after binding with albumin (Kessler et al. 1992; Kessler and Wolfbeis 1992). These two dyes, however, are not stable and hence their practical applications are limited. Later a derivative, AB 580, was developed to overcome the stability issue (Kessler et al. 1997a). These dyes are capable of highly specific binding with albumin to undergo strong fluorescence enhancement and are not subject to interference by other urinary proteins with minimum sample pretreatment (Kessler et al. 1997b). The AB 580-based assay was also applied to a glass microchip equipped with a fluorescence microscope for detection of albumin. However, the limit of detection (LOD) was not determined and further clinical applications were not performed (Kamholz et al. 1999). Recently, this AB 580-based assay was performed on a disposable microchip with an integrated fluorescence detection system based on thin-film, organic, light emitting diodes. A linear range of 10–100 mg/L was obtained (Hofmann et al. 2005). Although the LOD obtained by this integrated microchip is sufficiently low enough for the determination of MAU, only one sample can be assayed in one single operation in this work. Moreover, clinical samples were not assayed.
In this study, we present a microchip integrated with a fan-shaped micropump and micromixers capable of determining the albumin concentrations of six clinical urine samples in one single operation using an AB 580-based assay. Due to the unique design of the microchip, only two electromagnetic valves (EMVs) are required for actuating the on-chip micropump and micromixers. We also compare the results from clinical urine samples processed by the microchip with those obtained by the clinically used immunoturbidimetric method (Thakkar et al. 1997), to evaluate the performance of the proposed microchip.
2 Materials and methods
2.1 Chemicals and reagents
Dextran sulfate (DS, MWav 5000), hexadimethrine bromide (Polybrene, PB), sodium hydroxide (NaOH) and ethanol were obtained from Sigma-Aldrich (Louis, USA). The poly(dimethylsiloxane) (PDMS) kit was purchased from Dow Corning Corp. (Midland, USA). For the casting process, the PDMS elastomer and the curing agent were mixed in a weight ratio of 10:1 and cured at 80 °C for 4 hours. The albumin fluorescence assay kit was purchased from Fluka (Buchs, Switzerland). The assay reagent was freshly prepared by mixing reagent A (solution of AB 580 in 2-propanol) with reagent B (buffer solution, pH 7.0 ± 0.2) in both a volume ratio of 1:50 or 1:100. The albumin stock solution was prepared by dissolving 10 mg of HSA in 5 mL of Milli-Q water, and the albumin standard solutions for establishing calibration curves were prepared from this stock solution using the diluted solution provided in the kit. For fluorescence detection, the samples or standard solutions (6 μL) were mixed and reacted with the assay reagent (30 μL) through the operation of the micropump and micromixers.
2.2 Chip design
The fan-shaped micropump consists of six liquid channels, six air chambers, resilient PDMS membrane structures and normally closed valves (NCVs). As shown in Fig. 1(c), the NCV of the micropump is a PDMS-based floating block structure located inside the liquid channel, and is activated by hydraulic pressure generated by the motion of the PDMS membrane (Yang et al. 2009a). The NCV is used to prevent the backflow of the liquid during the pumping (Fig. 1(d-1)) and mixing (Fig. 1(d-2)) steps.
The microchip integrates micromixers which utilize pneumatically-driven membranes to generate the swirling flow in mixing chambers. As shown in Figure (d-2), the micromixer consists of two layers of PDMS substrates and a glass substrate layer. The top PDMS substrate contains two air chambers with connecting air channels. The width and height of the connecting air channels are 1,000 μm and 300 μm, respectively. The bottom PDMS substrate is comprised of a mixing chamber with two liquid channels. When compressed air is supplied to the two air chambers through the connecting air channels, the PDMS membranes are deflected sequentially and generate the swirling flow inside the circular mixing chamber. Meanwhile, the PDMS membrane above the NCV is also deflected to prevent the NCV from opening during the mixing step. Note that the connecting air channels are used to connect all the six micromixers so that only one EMV is required to activate the six micromixers in one single operation.
Prior to fluorescence detection, 6 μL of standard solutions or urine samples are first pipetted into each of the reservoirs and 190 μL of freshly prepared assay reagent is pipetted into the reagent well. The assay reagent is then pumped into the reservoirs (30 μL for each reservoir) through the fan-shaped micropump, followed by mixing with the standard solutions or samples in the reservoirs by the micromixers. After mixing, the compressed air is continuously applied to maintain the liquid level until the fluorescence detection is completed.
2.4 Experimental setup
Signals are detected on-chip via fluorescence detection. The detection system is constructed by modifying a commercial reflection microscope (model BX41, Olympus, Tokyo Japan) (Kuo et al. 2009). The light source radiating from a mercury lamp is filtered by a band-pass optical filter (540–580 nm) and is focused into a spot with a diameter of 3 mm on the reaction reservoir by a 50× (numerical aperture = 0.5) long-working-distance objective lens for exciting the AB 580 dyes. The emitted fluorescence is then collected by the same objective lens and is passed through a dichroic beam splitter (595 nm), a band-pass optical filter (600–660 nm), a pinhole with a diameter of 1.0 mm, and finally is detected by a photo-multiplier tube operating at 600 V (C3830, R928, Hamamatsu Photonics, Tokyo, Japan). Amplified photo-electronic signals are converted into digital signals and processed by a computer using a 24-bit commercial data acquisition interface (Model 9924–2; Scientific Information Service, Taipei, Taiwan).
3 Results and discussion
3.1 Surface modification
3.2 Characterization of the micropump
3.3 Characterization of the micromixer
3.4 Calibration curves
3.5 Clinical urine sample test
A comparison of throughput, detection limit, working range, and sample consumption between the prototype microchip and a conventional assay
Conventional assay a
2–40 mg/L b
This work presents a unique microfluidic-based platform capable of simultaneously determining low levels of albumin in six urine samples, in one single operation, based on a dye-binding reagent. This platform is less labor intensive, consumes less samples and reagents, and provides a fast analysis (114 sec for each operation). The experimental results are consistent with conventional immunoassays. Moreover, due to the low reagent price (no immunological reagents are required) and the excellent performance of the microchip, this platform may be used as a substitute for clinical immunoassays for the determination of MAU.
The authors gratefully acknowledge the financial support provided to this study by the National Science Council in Taiwan (NSC 97–2120-M-006–007).
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