Flow-through Microdispenser Research Articles

Determination of pesticides by capillary chromatography and SERS detection using a novel Silver-Quantum dots “sponge” nanocomposite

In this work the at-line capillary-liquid chromatography-(microdispenser)-surface-enhanced Raman spectroscopy coupling was investigated and applied to the determination of pesticides. The use of a microdispenser combined with the use of a precise and reproducible surface enhanced Raman spectroscopy (SERS) substrate yielded a chromatographic detection system with excellent analytical properties. The microdispenser was coupled to a moving CaF2 hot (80°C) plate using a flow-through microdispenser interface to collect the microdrops. Ag-QD nanocomposites, which are highly reproducible thanks to their sponge-shaped structure, were used as substrate with which to measure the SERS spectra in each spot of the plate. The limits of detection ranged from 0.2 to 0.5ng of pesticide injected (chlortoluron, atrazine, diuron and terbuthylazine) and the precision ranged between 10.2 and 12.5%.

Determination of Pyrimidine and Purine Bases by Reversed-Phase Capillary Liquid Chromatography with At-Line Surface-Enhanced Raman Spectroscopic Detection Employing a Novel SERS Substrate Based on ZnS/CdSe Silver–Quantum Dots

We have developed a new SERS substrate based on the reduction of silver nitrate in the presence of ZnS-capped CdSe quantum dots. This substrate showed higher sensitivities as compared to a hydroxylamine-reduced silver sol. On the basis of this new substrate, at-line SERS detection was coupled with capillary liquid chromatography (cap-LC) for the separation and selective determination of pyrimidine and purine bases. For this purpose, wells of a dedicated microtiter plate were loaded with 20 μL of the SERS substrate and placed on an automated x,y translation stage. A flow-through microdispenser capable of ejecting 50 pL droplets, at a frequency 100 Hz, was used as the interface to connect the cap-LC system to the wells loaded with SERS substrate. A detailed study of the dependence of both the separation and the surface-enhanced Raman spectra of each base on the pH was performed to optimize the system for maximum sensitivity and selectivity. Highly satisfactory analytical figures of merit were obtained for the six investigated bases (cytosine, xanthine, hypoxanthine, guanine, thymine, and adenine) with detection limits ranging between 0.2 and 0.3 ng injected on the capillary LC column, and the precisions were in the range of 3.0-6.3%.

Capillary liquid chromatography with off-line mid-IR and Raman micro-spectroscopic detection: analysis of chlorinated pesticides at ppb levels

A flow-through microdispenser was used as a solvent elimination interface, allowing vibrational spectroscopic detection in capillary liquid chromatography in addition to standard UV detection. Using a flow-through microdispenser, robust and stable deposition of picoliter-sized droplets on a CaF(2) plate window was achieved. The CaF(2) window was placed on a thermostated sample holder (80 degrees C) mounted on a computerized x, y stage for achieving fast solvent evaporation and enabling recording of the chromatogram as a trace of deposited material. The dried residues that were formed had diameters of a few tens of micrometers and were analysed by mid-IR and Raman micro-spectroscopy. Conditions were optimized for high sensitivity of measurement and maintaining chromatographic resolution during the deposition step. Due to the destruction-free character of Raman and FTIR spectroscopy, these techniques could be applied sequentially to interrogate the same deposits. To test the usefulness of the methodology for environmental analysis, the determination and unambiguous identification of chlorinated pesticides (chlortoluron, diuron, atrazine, and terbuthylazine) in river water was used as an example, obtaining limits of identification of 2 ng analyte on-column and precision of approximately 10% RSD. The application of the developed method to spiked real river samples demonstrated the identification power of the proposed method as, in addition to the four previously studied pesticides, two additional pesticides (simazine and isoproturon) could also be detected and identified.

Containerless reaction monitoring in ionic liquids by means of Raman microspectroscopy

Reaction monitoring by Raman microspectroscopy in levitated room temperature ionic liquid (RTIL) droplets is reported. Due to their non-volatility, RTIL droplets are well-suited to act as wall-less microreactors. The droplets were produced by a piezoelectric flow-through microdispenser connected to an automated flow injection system and were levitated by an acoustic trap. Taking advantage of the flow system versatility, the sequence of reagents was easily changed to study a model organic reaction: the Knoevenagel condensation. The reaction was followed by Raman microspectrometry and the obtained spectra were analysed using multivariate curve resolution to retrieve the concentration profiles and pure spectra of reactants, intermediates and products involved in the reaction. In addition, information about solvation interactions was obtained by monitoring the desolvation process taking place when a volatile co-solvent evaporated from the droplet.

Flow-through microdispenser for interfacing μ-HPLC to Raman and mid-IR spectroscopic detection

A flow-through microdispenser has been coupled to a micro HPLC separation system and used as a solvent elimination interface for Fourier transform infrared (FTIR) and Raman spectroscopic detection of the separated compounds. Using the microdispenser picoliter sized droplets can be generated and deposited on an appropriate target placed on a computerized x, y-stage. Evaporation of volatile solvent and buffer is rapid and allows analysis of the obtained dry deposits by various techniques. Due to the destruction free character of Raman and FTIR spectroscopy they can be applied sequentially to interrogate the same deposit. In the reported application five phenolic acids typically present in wine have been separated on a C-18 column technique using a mixture of water, methanol and acetic acid as mobile phase. For spectrum acquisition infrared and Raman microscopes have been used. The spectra recorded from the dried deposits of the separated compounds agreed well with the reference spectra of corresponding components.

A new device for coating single particles under controlled conditions

A new device for coating a single levitated particle in a controllable environment is designed and tested. This enables fluidized bed processing to be simulated experimentally on a single-particle level. The device consists of a coating chamber, which contains a capillary tube for levitating the particle, a micro-dispenser for producing discrete drops of controlled size and velocity and a device for supplying gas with specified temperature and humidity. The coating chamber consists of two parts, a confined space where the particle is levitated and a droplet insertion cone where the coating solution is injected into the particle suspending gas flow. A capillary with a well-defined diameter connects the droplet insertion cone and the area where the particle is levitated. The device is equipped with a piezo-actuated flow-through micro-dispenser that has the ability to produce discrete droplets with high reproducibility in terms of droplet size and velocity. The gas required for the coating process is taken from a gas container where the water content is analysed and kept at a minimum. A liquid flow is then introduced into the gas flow at a well-defined flow rate, mixed and evaporated in a three-way mixing vault. The humidified gas flow is then split into two separate flows; a suspending gas flow and a protecting gas flow for the inside of the coating chamber. The device is equipped with a high-speed video camera for monitoring both droplet production and droplet impact. Temperatures and flow rates throughout the device are measured and logged. Preliminary results show the influence of solvent, gas quality and coating procedure on the quality of the coating.

On-line monitoring of airborne chemistry in levitated nanodroplets: in situ synthesis and application of SERS-active Ag-Sols for trace analysis by FT-Raman spectroscopy.

We report a new strategy for on-line monitoring of chemical reactions in ultrasonically levitated, nanoliter-sized droplets by Raman spectroscopy. A flow-through microdispenser connected to an automated flow injection system was used to dose picoliter droplets into the node of an ultrasonic trap. Taking advantage of the flow-through characteristics of the microdispenser and the versatility of the automated flow system, a well-defined sequence of reagents could be injected via the microdispenser into the levitated droplet placed in the focus of the collection optics of the Fourier transform Raman spectrometer. In that way, chemical reactions could be carried out and monitored on-line. The developed system was used for fast, reproducible, in situ synthesis of a highly active surface enhanced Raman scattering (SERS) sol resulting from the reduction of silver nitrate with hydroxylamine hydrochloride in basic conditions. With this chemical system, SERS substrate preparation could be achieved at room temperature and in short time. The in situ prepared silver sol was used for trace analysis of several organic test molecules that were injected into the levitated SERS-active droplet again using the microdispenser. The concentration dependence of the SERS spectra was studied using 9-aminoacridine, revealing that down to the femtogram region high-quality SERS spectra could be obtained. Additionally, SERS spectra of 6-mercaptopurine, thiamine, and acridine were recorded in the levitated drop as well.

Miniaturized flowthrough microdispenser with piezoceramic tripod actuation

In this paper, the further development of a silicon flowthrough microdispenser is described. Previously reported designs of the dispenser used bimorph, and later multilayered, piezoelectric actuator elements for the generation of droplets. The introduction of a multilayered actuator significantly reduced the voltage amplitude needed to dispense droplets. Dispenser properties relevant for chemical analysis systems, e.g., reduced sample volume, internal surface area, and dispersion, were improved by miniaturization of the device. A new actuator design, the tripod, is presented to enable further dispenser miniaturization and to facilitate device assembly. Tripod actuators were manufactured using a prototyping process, based on micromilling, for multilayer piezoceramic components. A building technique for miniaturized electrical interconnects, based on microstructured flexible printed circuits, is also suggested in line with the prospect of future miniaturization. The microfluidic properties of the tripod-actuated dispenser were evaluated. Stable droplet generation in the frequency range from 0 to 3 kHz was demonstrated, providing a maximum dispensed flow rate of 7.8 /spl mu/L/min.

Flow-through Picoliter Dispenser: A New Approach for Solvent Elimination in FT-IR Spectroscopy

A new interface for FT-IR analysis of liquid samples on the basis of solvent elimination is presented. The approach is based on a piezoactuated flow-through microdispenser, a device built of two microstructured silicon wafers designed for micro-liquid handling. It could be verified during preliminary studies using a sequential injection (SI) system for automated liquid handling that the flow-through microdispenser as a possible interface for flow system–FT-IR analysis has the capability of meeting the demands of hyphenated miniaturized liquid handling systems (e.g., μ-HPLC, microhigh performance liquid chromatography), as it successfully provides highly stable, reliable and reproducible operating conditions for liquid handling in the picoliter range. Moreover, an increase in sensitivity for FT-IR measurements could be achieved, lowering the mass detection limit of sugars (such as the investigated sucrose) to 53 picograms. As is demonstrated on the example of an HPLC separation of a mixture of glucose and fructose, interfacing LC systems to FT-IR using a piezoactuated flow-through microdispenser is a feasible and promising approach.

Ready-made matrix-assisted laser desorption/ionization target plates coated with thin matrix layer for automated sample deposition in high-density array format.

The methodology for ready-made matrix-assisted laser desorption/ionization (MALDI) target plates covered with an optimized thin layer consisting of matrix and nitrocellulose has been developed. Piezoelectric microdispensing enabled sample depositions in a high-density array format of 2000 sample depositions on a conventionally sized target plate (45 x 47 mm). The sample depositions were made reproducibly in a fully automated mode by using an in-house developed computer-controlled piezoelectric flow-through microdispenser. Additionally, the piezoelectric technique facilitated significant analyte enrichment that increased the detection sensitivity. The MS signal was obtained rapidly, generally within ten laser pulses. An airbrush device was used to generate a fine spray of matrix and nitrocellulose dissolved in acetone. The acetone evaporated instantly when reaching the target plate leaving the entire surface with a thin and uniform matrix/nitrocellulose coating consisting of very small crystals of matrix embedded in the nitrocellulose. These crystals acted as a seed-layer on subsequent analyte depositions, rendering homogeneous sample spots when using alpha-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The relative standard deviation of the signal intensity between spots was (20-30)% (n = 30). The detection sensitivity was improved by restricting the sample spot diameter to 300 microm. The spot size was affected by the deposition rate and the evaporation rate of the dispensed sample volume. Mass spectra of a 25-amol peptide mixture deposition were successfully recorded.

A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser.

Micrometer-sized enzyme grids were fabricated on gold surfaces using a novel method based on a flow-through microdispenser. The method involves dispensing very small droplets of enzyme solution (approximately 100 pL) during the concomitant relative movement of a gold substrate with respect to the nozzle of a microdispenser, resulting in enzyme patterns with a line width of approximately 100 microm. Different immobilization methods have been evaluated, yielding either enzyme monolayers using functionalized self-assembled thiol monolayers for covalent binding of the enzyme or enzyme multilayers by cross-linking or entrapping the enzymes in a polymer film. The latter immobilization techniques allow the formation of coupled multienzyme structures. On the basis of this feature, coupled bienzyme (glucose oxidase and catalase) or three-enzyme (alpha-glucosidase, mutarotase, and glucose oxidase) microstructures consisting of line patterns of one enzyme intersecting with the patterned lines of the other enzyme(s) were fabricated. By means of scanning electrochemical microscopy (SECM) operated in the generator-collector mode, the enzyme microstructures and their integrity were visualized using the localized detection of enzymatically produced/consumed H2O2. A calibration curve for glucose could be obtained by subsequent SECM line scans over a glucose oxidase microstructure for increasing glucose concentrations, demonstrating the possibility of obtaining localized quantitative data from the prepared microstructures. Possible applications of these enzyme microstructures for multianalyte detection and interference elimination and for screening of different biosensor configurations are highlighted.

Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry

An integrated protein microcharacterization/identification platform has been developed. The system has been designed to allow a high flexibility in order to tackle challenging analytical problems. The platform comprises a cooled microautosampler, an integrated system for microcolumn HPLC, and a capillary reversed-phase column that is interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system via a low internal volume flow-through microdispenser. The chromatographic separation is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and re-analysis of interesting fractions. The use of target plates pre-coated with matrix simplified and increased the robustness of the system. By including a separation step prior to the MALDI-TOF-MS analysis and hereby minimizing suppression effects allowed us to obtain higher sequence coverage of proteins compared to conventional MALDI sample preparation methodology. Additionally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485–496 (ALGADDSYYTAR) of tyrosine kinase ZAP-70 were identified at sensitivities reaching 150 amol.

Capillary liquid chromatography interfaced to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an on-line coupled piezoelectric flow-through microdispenser

A piezoelectric flow-through microdispenser interfacing capillary liquid chromatography (LC) with matrix-assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-TOF MS) was developed for the identification of biomolecules. The MALDI target plate was placed on a computer controlled high-resolution x-y stage, on to which the column effluent was deposited as discrete spots, which thereby facilitated tracing of the chromatographic separation. The entire target plate was sprayed with a homogeneous layer of alpha-cyano-4-cinnamic acid mixed with nitrocellulose by using an air-brush. Hence the tedious manual handling of a micropipetter applying matrix solution on top of each fraction collected spot was avoided. The pre-made target plates were stable for at least 3 weeks if kept in darkness at room temperature, which easily allowed re-analysis of dispensed sample spots. The integrated microsystem was characterized and optimized by means of fluidics, dispersion, operational stability and sensitivity parameters. The dispensing unit was developed specifically to match high-resolution capillary LC separations using a dispenser with an internal volume from inlet to the ejecting nozzle of 250 nl. Minimizing dead volumes was crucial in order to maintain the chromatographic resolution. The volume of the ejected droplets was of the order of 60 pl. Successful separations of seven immunoregulating peptides were made: ACTH 1-17, bradykinin, enkephalin, angiotensin III, angiotensin II, angiotensin I and ACTH 18-39. On-line sample dispensing on the target plate in combination with trace enrichment followed by automated MALDI-TOF MS identification is demonstrated, reaching a sensitivity of 100 amol.

Design and development of a silicon microfabricated flow-through dispenser for on-line picolitre sample handling

The development of a piezoactuated flow-through microdispenser is described. The dispenser can be used for on-line sampling in a continuous-liquid-flow system. A major application area is rapid sample handling in chemical microsystems, e.g. automated high-throughput analysis or screening systems. The microdispenser is constructed of two joined silicon structures forming a flow-through channel. One channel wall couples the impulse movement of a piezoceramic element into the flow channel, generating a pressure pulse. Droplets (of typically 100 picolitres) are ejected from an orifice in the opposite channel wall. The sequential improvements of the dispenser over three generations are presented. The actuation voltage could be lowered from 150 V to approximately 50 V. The final version of the dispenser had an internal volume of 2.6 microlitres. The impact on droplet stability and directivity of introducing a protruding p-n etch-stop-defined nozzle and a means of matching surface properties to the dispensed liquid for improved performance are discussed. The maximum frequency for stable droplet formation was found to be approximately 500 Hz.

Sample preparation strategies for low-volume determinations inIn vitro studies of proteins by use of MALDI-TOF MS

A flow-through microdispenser, manufactured from micromachined silicon and based on a piezoelectric element, has been used as an interface for high-density target sample deposition in matrix assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF). The dispenser has been used for sample preparation in MALDI-TOF MS analysis proteins. Single 110-picoliter sample droplets containing between 200 and 300 attomol proteins can be determined. The sensitivity of analysis of a dilute protein was increased by use of pre-concentration procedure in which the protein solution was dispensed as four sequences each of 200 droplets.