Continuous Flow with Reagent Injection on an Inlaid Microfluidic Platform Applied to Nitrite Determination.

A continuous flow with reagent injection method on a novel inlaid microfluidic platform for nitrite determination has been successfully developed. The significance of the high-frequency monitoring of nutrient fluctuations in marine environments is crucial for understanding our impacts on the ecosystem. Many in-situ systems face limitations in high-frequency data collection and have restricted deployment times due to high reagent consumption. The proposed microfluidic device employs automatic colorimetric absorbance spectrophotometry, using the Griess assay for nitrite determination, with minimal reagent usage. The sensor incorporates 10 solenoid valves, four syringes, two LEDs, four photodiodes, and an inlaid microfluidic technique to facilitate optical measurements of fluid volumes. In this flow system, Taylor-Aris dispersion was simulated for different injection volumes at a constant flow rate, and the results have been experimentally confirmed using red food dye injection into a carrier stream. A series of tests were conducted to determine a suitable injection frequency for the reagent. Following the initial system characterization, seven different standard concentrations ranging from 0.125 to 10 µM nitrite were run through the microfluidic device to acquire a calibration curve. Three different calibrations were performed to optimize plug length, with reagent injection volumes of 4, 20, and 50 µL. A straightforward signal processing method was implemented to mitigate the Schlieren effect caused by differences in refractive indexes between the reagent and standards. The results demonstrate that a sampling frequency of at least 10 samples per hour is achievable using this system. The obtained attenuation coefficients exhibited good agreement with the literature, while the reagent consumption was significantly reduced. The limit of detection for a 20 µL injection volume was determined to be 94 nM from the sample intake, and the limit of quantification was 312 nM. Going forward, the demonstrated system will be packaged in a submersible enclosure to facilitate in-situ colorimetric measurements in marine environments.

Compact and automated eDNA sampler for in situ monitoring of marine environments.

Using environmental DNA (eDNA) to monitor biodiversity in aquatic environments is becoming an efficient and cost-effective alternative to other methods such as visual and acoustic identification. Until recently, eDNA sampling was accomplished primarily through manual sampling methods; however, with technological advances, automated samplers are being developed to make sampling easier and more accessible. This paper describes a new eDNA sampler capable of self-cleaning and multi-sample capture and preservation, all within a single unit capable of being deployed by a single person. The first in-field test of this sampler took place in the Bedford Basin, Nova Scotia, Canada alongside parallel samples taken using the typical Niskin bottle collection and post-collection filtration method. Both methods were able to capture the same aquatic microbial community and counts of representative DNA sequences were well correlated between methods with R[Formula: see text] values ranging from 0.71-0.93. The two collection methods returned the same top 10 families in near identical relative abundance, demonstrating that the sampler was able to capture the same community composition of common microbes as the Niskin. The presented eDNA sampler provides a robust alternative to manual sampling methods, is amenable to autonomous vehicle payload constraints, and will facilitate persistent monitoring of remote and inaccessible sites.

An Automated Microfluidic Analyzer for In Situ Monitoring of Total Alkalinity.

We have designed, built, tested, and deployed an autonomous in situ analyzer for seawater total alkalinity. Such analyzers are required to understand the ocean carbon cycle, including anthropogenic carbon dioxide (CO2) uptake and for mitigation efforts via monitoring, reporting, and verification of carbon dioxide removal through ocean alkalinity enhancement. The microfluidic nature of our instrument makes it relatively lightweight, reagent efficient, and amenable for use on platforms that would carry it on long-term deployments. Our analyzer performs a series of onboard closed-cell titrations with three independent stepper-motor driven syringe pumps, providing highly accurate mixing ratios that can be systematically swept through a range of pH values. Temperature effects are characterized over the range 5-25 °C allowing for field use in most ocean environments. Each titration point requires approximately 170 µL of titrant, 830 µL of sample, 460 J of energy, and a total of 105 s for pumping and optical measurement. The analyzer performance is demonstrated through field data acquired at two sites, representing a cumulative 25 days of operation, and is evaluated against laboratory measurements of discrete water samples. Once calibrated against onboard certified reference material, the analyzer showed an accuracy of -0.17 ± 24 µmol kg-1. We further report a precision of 16 µmol kg-1, evaluated on repeated in situ measurements of the aforementioned certified reference material. The total alkalinity analyzer presented here will allow measurements to take place in remote areas over extended periods of time, facilitating affordable observations of a key parameter of the ocean carbon system with high spatial and temporal resolution.

A submersible phosphate analyzer for marine environments based on inlaid microfluidics.

In situ sensors are needed to further our understanding of phosphate flux dynamics in marine environments during short term events such as tidal cycles, algae blooms and runoff periods. Here, we present a fully automated in situ phosphate analyzer based on an inlaid microfluidic absorbance cell technology. The microfluidic device employs colorimetric absorbance spectrophotometry, using the phosphomolybdenum blue (PMB) assay modified by the addition of polyvinylpyrrolidone (PVP), to measure phosphate concentrations in seawater. Bench top calibrations were performed with both copper(II) sulfate dye and the PMB assay, as well as temperature sensitivity studies to characterize the sensor's performance in a range of conditions. It achieves a limit of detection of 15.2 nM, a limit of quantification of 50.8 nM, and a high in situ precision with a relative standard deviation of less than 1.5% across three consecutive measurements. Two consecutive field deployments are conducted as assessments for its intended in situ applications. The sensor is first deployed from a pier at a depth of 6 m, with simultaneous bottle samples taken to perform cross-validation. It is next deployed on the Stella Maris testbed, a multi-sensor seabed platform (MSSP), 100 m offshore and 9 m deep in the inlet to the Bedford Basin in Nova Scotia, Canada. Over 300 successful phosphate measurements were acquired, showing the influence of the tidal cycle, and confirming the sensor's viability in observing nutrient flux dynamics with nanomolar variations.

Simultaneous Absorbance and Fluorescence Measurements Using an Inlaid Microfluidic Approach.

A novel microfluidic optical cell is presented that enables simultaneous measurement of both light absorbance and fluorescence on microlitre volumes of fluid. The chip design is based on an inlaid fabrication technique using clear and opaque poly(methyl methacrylate) or PMMA to create a 20.2 mm long optical cell. The inlaid approach allows fluid interrogation with minimal interference from external light over centimeter long path lengths. The performance of the optical cell is evaluated using a stable fluorescent dye: rhodamine B. Excellent linear relationships (R2 > 0.99) are found for both absorbance and fluorescence over a 0.1-10 µM concentration range. Furthermore, the molar attenuation spectrum is accurately measured over the range 460-550 nm. The approach presented here is applicable to numerous colorimetric- or fluorescence-based assays and presents an important step in the development of multipurpose lab-on-chip sensors.

Airy beams on incoherent background.

We present a class of diffraction-free partially coherent beams, each member of which comprises a finite-power, non-accelerating Airy bump residing on a statistically homogeneous, Gaussian-correlated background. We examine free-space propagation of soft apertured realizations of the proposed beams and show that their evolution is governed by two spatial scales: the coherence width of the background and the aperture size. A relative magnitude of these factors determines the practical range of propagation distances over which the novel beams can withstand diffraction. The proposed beams can find applications to imaging and optical communications through random media.

An Energy Efficient Thermally Regulated Optical Spectroscopy Cell for Lab-on-Chip Devices: Applied to Nitrate Detection.

Reagent-based colorimetric analyzers often heat the fluid under analysis for improved reaction kinetics, whilst also aiming to minimize energy use per measurement. Here, a novel method of conserving heat energy on such microfluidic systems is presented. Our design reduces heat transfer to the environment by surrounding the heated optical cell on four sides with integral air pockets, thereby realizing an insulated and suspended bridge structure. Our design was simulated in COMSOL Multiphysics and verified in a polymethyl methacrylate (PMMA) device. We evaluate the effectiveness of the insulated design by comparing it to a non-insulated cell. For temperatures up to 55 °C, the average power consumption was reduced by 49.3% in the simulation and 40.2% in the experiment. The designs were then characterized with the vanadium and Griess reagent assay for nitrate at 35 °C. Nitrate concentrations from 0.25 µM to 50 µM were tested and yielded the expected linear relationship with a limit of detection of 20 nM. We show a reduction in energy consumption from 195 J to 119 J per 10 min measurement using only 4 µL of fluid. Efficient heating on-chip will have broad applicability to numerous colorimetric assays.

A Magnetically Tunable Check Valve Applied to a Lab-on-Chip Nitrite Sensor.

Presented here is the fabrication and characterization of a tunable microfluidic check valve for use in marine nutrient sensing. The ball-style valve makes use of a rare-earth permanent magnet, which exerts a pulling force to ensure it remains passively sealed until the prescribed cracking pressure is met. By adjusting the position of the magnet, the cracking pressure is shown to be customizable to meet design requirements. Further applicability is shown by integrating the valve into a poly(methyl methacrylate) (PMMA) lab-on-chip device with an integrated optical absorbance cell for nitrite detection in seawater. Micro-milling is used to manufacture both the valve and the micro-channel structures. The valve is characterized up to a flow rate of 14 mL min-1 and exhibits low leakage rates at high back pressures (<2 µL min-1 at ~350 kPa). It is low cost, requires no power, and is easily implemented on microfluidic platforms.

Determination of boron concentration in oilfield water with a microfluidic ion exchange resin instrument.

We developed and validated a microfluidic instrument for interference-free determination of boron in produced water. The instrument uses a boron-specific chelating resin to separate the analyte from its complex matrix. Ten produced water samples were analyzed with the instrument and the results were successfully validated against ICP-MS measurements. Removing interference effects enables precise boron measurement for wastewater even with high total dissolved solid (TDS) levels. 1,4-Piperazinediethanesulfonic acid conditions the resin and maintains the optimum pH for boron adsorption from the sample. Boron is then eluted from the resin using a 10% sulfuric acid solution and its concentration measured with the colorimetric carminic acid assay in 95% sulfuric acid. The use of a microfluidic mixer greatly enhances the sensitivity and kinetics of the carminic acid assay, by factors of 2 and 7.5, respectively, when compared against the same assay performed manually. A maximum sensitivity of 2.5mg(-1)L, a precision of 4.2% over the 0-40.0mgL(-1) measuring range, a 0.3mgL(-1) limit of detection, and a sampling rate of up to four samples per hour were achieved. Automation and microfluidics reduce the operator workload and fluid manipulation errors, translating into safer and higher-quality measurements in the field.

Determination of boron in produced water using the carminic acid assay.

Using the carminic acid assay, we determined the concentration of boron in oilfield waters. We investigated the effect of high concentrations of salts and dissolved metals on the assay performance. The influence of temperature, development time, reagent concentration, and water volume was studied. Ten produced and flowback water samples of different origins were measured, and the method was successfully validated against ICP-MS measurements. In water-stressed regions, produced water is a potential source of fresh water for irrigation, industrial applications, or consumption. Therefore, boron concentration must be determined and controlled to match the envisaged waste water reuse. Fast, precise, and onsite measurements are needed to minimize errors introduced by sample transportation to laboratories. We found that the optimum conditions for our application were a 5:1 mixing volume ratio (reagent to sample), a 1 g L(-1) carminic acid concentration in 99.99% sulfuric acid, and a 30 min reaction time at ambient temperature (20 °C to 23 °C). Absorption values were best measured at 610 nm and 630 nm and baseline corrected at 865 nm. Under these conditions, the sensitivity of the assay to boron was maximized while its cross-sensitivity to dissolved titanium, iron, barium and zirconium was minimized, alleviating the need for masking agents and extraction methods.

Asphaltenes yield curve measurements on a microfluidic platform.

We describe a microfluidic apparatus and method for performing asphaltene yield measurements on crude oil samples. Optical spectroscopy measurements are combined with a microfluidic fluid handling platform to create an automated microfluidic apparatus to measure the asphaltene yield. The microfluidic measurements show good agreement with conventional wet chemistry measurements as well as available models. The initial absorbance of the oil is measured, and asphaltenes are removed from the oil by the gradual addition of n-alkane, which leads to flocculation and subsequent filtration. The absorbance of the de-asphalted oil (maltenes) is then measured and the initial asphaltene content is determined by the change in absorbance. The solubility of asphaltene is evaluated by varying the titrant-to-oil ratio (e.g., n-heptane-oil), which induces no, partial, or full precipitation of asphaltenes depending on the chosen ratio. The absorbance of the filtrate is measured and normalized to the maximum content to determine the fractional precipitation at each ratio. Traditionally, a yield curve comprised of 20 such ratios would require weeks to months to generate, while consuming over 6 L of solvent and more than 100 g of crude oil sample. Using the microfluidic approach described here, the same measurement can be performed in 1 day, with 0.5 L of solvent and 10 g of crude oil sample. The substantial reduction in time and consumables will enable more frequent asphaltene yield measurements and reduce its environmental impact significantly.

A high performance microfluidic analyser for phosphate measurements in marine waters using the vanadomolybdate method.

We report a high performance autonomous analytical system based on the vanadomolybdate method for the determination of soluble reactive phosphorus in seawater. The system combines a microfluidic chip manufactured from tinted poly (methyl methacrylate) (PMMA), a custom made syringe pump, embedded control electronics and on-board calibration standards. This "lab-on-a-chip" analytical system was successfully deployed and cross-compared with reference analytical methods in coastal (south west England) and open ocean waters (tropical North Atlantic). The results of the miniaturized system compared well with a reference bench-operated phosphate auto-analyser and showed no significant differences in the analytical results (student's t-test at 95% confidence level). The optical technology used, comprising of tinted PMMA and polished fluidic channels, has allowed an improvement of two orders of magnitude of the limit of detection (52 nM) compared to currently available portable systems based on this method. The system has a wide linear dynamic range 0.1-60 µM, and a good precision (13.6% at 0.4 µM, n=4). The analytical results were corrected for silicate interferences at 0.7 µM, and the measurement frequency was configurable with a sampling throughput of up to 20 samples per hour. This portable micro-analytical system has a low reagent requirement (340 µL per sample) and power consumption (756 J per sample), and has allowed accurate high resolution measurements of soluble reactive phosphorus in seawater.

Measurement of asphaltenes using optical spectroscopy on a microfluidic platform.

We present a microfluidic apparatus and method for the measurement of asphaltene content in crude-oil samples. The measurement is based on an optical absorption technique, where it was established that asphaltene coloration correlated linearly with asphaltene weight content. The initial absorbance of the oil is measured, and asphaltenes are removed from the oil by the addition of n-alkane, leading to flocculation and subsequent filtration. The absorbance of the deasphalted oil (maltenes) is then measured, and the initial asphaltene content is revealed by the change in absorbance. The asphaltene optical densities correlated linearly with conventional weight measurement results (e.g., ASTM D6560) for 38 crude-oil samples from around the world. Sample measurement repeatability was shown to be within ±2% over several months. Other aspects influencing performance of the system were evaluated, including plug dispersion, flocculation kinetics, membrane degradation, and channel clogging. The microfluidic approach described here permits asphaltene content measurement in less than 30 min as opposed to days required with traditional gravimetric techniques. This many-fold reduction in measurement time will enable more frequent characterization of crude oil samples.

Lab-on-chip measurement of nitrate and nitrite for in situ analysis of natural waters.

Microfluidic technology permits the miniaturization of chemical analytical methods that are traditionally undertaken using benchtop equipment in the laboratory environment. When applied to environmental monitoring, these "lab-on-chip" systems could allow high-performance chemical analysis methods to be performed in situ over distributed sensor networks with large numbers of measurement nodes. Here we present the first of a new generation of microfluidic chemical analysis systems with sufficient analytical performance and robustness for deployment in natural waters. The system detects nitrate and nitrite (up to 350 µM, 21.7 mg/L as NO(3)(-)) with a limit of detection (LOD) of 0.025 µM for nitrate (0.0016 mg/L as NO(3)(-)) and 0.02 µM for nitrite (0.00092 mg/L as NO(2)(-)). This performance is suitable for almost all natural waters (apart from the oligotrophic open ocean), and the device was deployed in an estuarine environment (Southampton Water) to monitor nitrate+nitrite concentrations in waters of varying salinity. The system was able to track changes in the nitrate-salinity relationship of estuarine waters due to increased river flow after a period of high rainfall. Laboratory characterization and deployment data are presented, demonstrating the ability of the system to acquire data with high temporal resolution.

Evanescent photosynthesis: exciting cyanobacteria in a surface-confined light field.

The conversion of solar energy to chemical energy useful for maintaining cellular function in photosynthetic algae and cyanobacteria relies critically on light delivery to the microorganisms. Conventional direct irradiation of a bulk suspension leads to non-uniform light distribution within a strongly absorbing culture, and related inefficiencies. The study of small colonies of cells in controlled microenvironments would benefit from control over wavelength, intensity, and location of light energy on the scale of the microorganism. Here we demonstrate that the evanescent light field, confined near the surface of a waveguide, can be used to direct light into cyanobacteria and successfully drive photosynthesis. The method is enabled by the synergy between the penetration depth of the evanescent field and the size of the photosynthetic bacterium, both on the order of micrometres. Wild type Synechococcus elongatus (ATCC 33912) cells are exposed to evanescent light generated through total internal reflection of red (λ = 633 nm) light on a prism surface. Growth onset is consistently observed at intensity levels of 79 ± 10 W m(-2), as measured 1 µm from the surface, and 60 ± 8 W m(-2) as measured by a 5 µm depthwise average. These threshold values agree well with control experiments and literature values based on direct irradiation with daylight. In contrast, negligible growth is observed with evanescent light penetration depths less than the minor dimension of the rod-like bacterium (achieved at larger light incident angles). Collectively these results indicate that evanescent light waves can be used to tailor and direct light into cyanobacteria, driving photosynthesis.

Nanomolar detection with high sensitivity microfluidic absorption cells manufactured in tinted PMMA for chemical analysis.

We describe a novel, cost effective and simple technique for the manufacture of high sensitivity absorption cells for microfluidic analytical systems. The cells are made from tinted polymethyl methacrylate (PMMA) in which microfluidic channels are fabricated. Two windows (typically 250 µm thick, resulting in little optical power loss) are formed at either end of the channel through which light is coupled. Unwanted stray light from the emitter passes through a greater thickness of the tinted substrate (typically the length of the cell) and is preferentially absorbed. In effect, this creates a pin-hole configuration over the length of the absorption cell, providing improved performances (sensitivity, S/N ratios, baseline noise and limit of detection) when used as an absorption cell compared to clear substrates. The method is used to achieve a LOD of 20 nM with a colourimetric iron assay and a LOD of 0.22 milli-absorption units with a pH assay.

An integrated microfluidic chip for chromosome enumeration using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful technique for probing the genetic content of individual cells at the chromosomal scale. Conventional FISH techniques provide a sensitive diagnostic tool for the detection of chromosomal alterations on a cell-by-cell basis; however, the cost-per-test in terms of reagent and highly qualified labour has prevented its wide-spread utilization in clinical settings. Here, we address the inefficient use of labour with the first integrated and automated on-chip FISH implementation, one that requires only minutes of setup time from the technician. Our microfluidic chip has lowered the reagent use by 20-fold, decreased the labour time by 10-fold, and substantially reduced the amount of support equipment needed. We believe this cost-effective platform will make sensitive FISH techniques more accessible for routine clinical usage.

Rapid on-chip postcolumn labeling and high-resolution separations of DNA.

When performing genetic analysis on microfluidic systems, labeling the sample DNA for detection is a critical preparation step. Labeling procedures often involve fluorescently tagged primers and PCRs, which lengthen experimental run times and introduce higher levels of complexity, increasing the overall cost per analysis. Alternatively, on-chip labeling techniques based on intercalating dyes permit rapid labeling of DNA fragments. However, as noted in the literature, the stochastic nature of dye-DNA complex formation hinders the native electrophoretic migration of DNA fragments, degrading the separation resolution. In this study, we present a novel method of controllably labeling DNA fragments at the end of the electrophoretic separation channel in a glass microfluidic chip. Permitting the DNA to separate and labeling just before detection, achieves the rapid labeling associated with intercalators while maintaining the high resolution of native DNA separations. Our analyses are completed in minutes, rather than the hours typical of sample prelabeling. We demonstrate an electrophoretic microchip-based intercalator labeling technique that achieves higher resolution performance than reported in the literature to date.