Development of a microfluidic paper-based analytical device for the determination of salivary aldehydes.

A low cost, disposable and easy to use microfluidic paper-based analytical device (µPAD) was developed for simple and non-invasive determination of total aldehydes in saliva with a potential to be used in epidemiological studies to assess oral cancer risk. The µPAD is based on the colour reaction between aldehydes (e.g. acetaldehyde, formaldehyde), 3-methyl-2-benzothiazolinone hydrazone (MBTH) and iron(III) to form an intense blue coloured formazan dye. The newly developed µPAD has a 3D design with two overlapping paper layers. The first layer comprises 15 circular detection zones (8 mm in diameter), each impregnated with 8 µL of MBTH, while the second layer contains 15 reagent zones (4 mm in diameter). Two µL of iron(III) chloride are added to each one of the second layer zones after the addition of sample to the detection zones in the first layer. All hydrophilic zones of the µPAD are defined by wax printing using a commercial wax printer. Due to the 2-step nature of the analytical reaction, the two paper layers are separated by a cellulose acetate interleaving sheet to allow for the reaction between the aldehydes in the saliva sample with MBTH to proceed first with the formation of an azine, followed by a blue coloured reaction between the azine and the oxidized by iron(III) form of MBTH, produced after the removal of the interleaving sheet. After obtaining a high resolution image of the detection side zone of the device using a flatbed scanner, the intensity of the blue colour within each detection zone is measured with Image J software. Under optimal conditions, the µPAD is characterised by a working range of 20.4-114.0 µM, limit of detection of 6.1 µM, and repeatability, expressed as RSD, of less than 12.7% (n = 5). There is no statistically significant difference at the 95% confidence level between the results obtained by the µPAD and the reference method (Student's t-test: 0.090 < 0.38). The optimized µPAD is stable for more than 41 days when stored in a freezer (≤-20 °C).

Determination of salivary cotinine through solid phase extraction using a bead-injection lab-on-valve approach hyphenated to hydrophilic interaction liquid chromatography.

Cotinine, the first metabolite of nicotine, is often used as a biomarker in the monitoring of environmental tobacco smoke (ETS) exposure due to its long half-life. This paper reports on the development of an at-line automatic micro-solid phase extraction (µSPE) method for the determination of salivary cotinine followed by its analysis via hydrophilic interaction liquid chromatography (HILIC). The SPE methodology is based on the bead injection (BI) concept in a mesofluidic lab-on-valve (LOV) flow system to automatically perform all SPE steps. Three commercially available reversed-phase sorbents were tested, namely, Oasis HLB, Lichrolut EN and Focus, and the spherically shaped sorbents (i.e., Oasis HLB and Focus) provided better packing within the SPE column and hence higher column efficiency. An HILIC column was chosen based on its potential for achieving higher sensitivity and better retention of polar compounds such as cotinine. The method uses an isocratic program with acetonitrile:100mM ammonium acetate buffer, pH 5.8 in 95:5 v/v ratio as the mobile phase at a flow rate of 1.0 mL min(-1). Using this approach, the linear calibration range was from 10 to 1000 ng which corresponded to 5-500 µg L(-1). The corresponding µSPE-BI-LOV system was proven to be reliable in the handing and analysis of viscous biological samples such as saliva, achieving a sampling rate of 6h(-1) and a limit of detection and quantification of 1.5 and 3µgL(-1), respectively.

Determination of acetaldehyde in saliva by gas-diffusion flow injection analysis.

The consumption of ethanol is known to increase the likelihood of oral cancer. In addition, there has been a growing concern about possible association between long term use of ethanol-containing mouthwashes and oral cancer. Acetaldehyde, known to be a carcinogen, is the first metabolite of ethanol and it can be produced in the oral cavity after consumption or exposure to ethanol. This paper reports on the development of a gas-diffusion flow injection method for the online determination of salivary acetaldehyde by its colour reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and ferric chloride. Acetaldehyde samples and standards (80 µL) were injected into the donor stream containing NaCl from which acetaldehyde diffused through the hydrophobic Teflon membrane of the gas-diffusion cell into the acceptor stream containing the two reagents mentioned above. The resultant intense green coloured dye was monitored spectrophotometrically at 600 nm. Under the optimum working conditions the method is characterized by a sampling rate of 9h(-1), a linear calibration range of 0.5-15 mg L(-1) (absorbance=5.40×10(-2) [acetaldehyde, mg L(-1)], R(2)=0.998), a relative standard deviation (RSD) of 1.90% (n=10, acetaldehyde concentration of 2.5 mg L(-1)), and a limit of detection (LOD) of 12.3 µg L(-1). The LOD and sampling rate of the proposed method are superior to those of the conventional gas chromatographic (GC) method (LOD=93.0 µg L(-1) and sampling rate=4 h(-1)). The reliability of the proposed method was illustrated by the fact that spiked with acetaldehyde saliva samples yielded excellent recoveries (96.6-101.9%), comparable to those obtained by GC (96.4-102.3%) and there was no statistically significant difference at the 95% confidence level between the two methods when non-spiked saliva samples were analysed.