Electrospray device for coupling microscale separations and other miniaturized devices with electrospray mass spectrometry

A miniaturized ion sprayer device is described which is suitable for coupling with chip-based analytical separation devices, multiwell plates, or surfaces containing residues of prepared samples. Two versions of a similar device are described. A "microsprayer" device suitable for coupling to the terminal edge of a capillary electrophoresis (CE) chip is constructed from modified 1/16-in. HPLC fittings. This microsprayer employs a free-standing liquid junction formed via continuous delivery of a flow (2-6 microL/min) of suitable solvent which carries the CE effluent through a pneumatically assisted electrospray (ion spray) needle positioned in front of an atmospheric pressure ionization (API) mass spectrometer. A related but larger "minisprayer" device is also described which employs the same features as the microsprayer, but with an extended sampling capillary tube which can reach into the depths of 96-, 384-, and 1536-multiwell plates containing either sample solutions or dried sample residues. The minisprayer may be positioned in front of an API ion sampling orifice and the multiwell plate positioned stepwise from sample to sample for analysis of trace samples contained in the wells. The resulting infusion-ion spray mass spectrometric analyses can provide sequential analysis of previously prepared biological samples containing small drug compounds, proteins, and related compounds. This same device is also shown to be useful for sampling from a surface containing trace level compounds of biological interest. Results are shown that demonstrate microscale separations and selected ion monitoring (SIM) capillary electrophoresis/mass spectrometry (CE/MS) detection of berberine and palmatine using the microsprayer. SIM ion spray determination of a 2 ng/microL solution of berberine contained as a dry residue in the bottom of a 384-well plate as well as full-scan electrospray mass spectra for low-picomole levels of cytochrome c contained in a 1536-well microtiter plate are shown. The respective micro- and minisprayer devices provide a simple yet effective means of transferring trace-level samples either from a microscale or chip-based separation device as well as samples contained in multiwell plates which are increasingly employed in high-throughput applications in the pharmaceutical industry.     

Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.     

Phase-changing sacrificial materials for solvent bonding of high-performance polymeric capillary electrophoresis microchips

A new method for solvent bonding polymeric substrates to form microfluidic systems has been demonstrated. Prior to device sealing, channels in an embossed poly(methyl methacrylate) (PMMA) piece are filled with a heated liquid (paraffin wax) that forms a solid sacrificial layer at room temperature. The sacrificial material prevents the bonding solvent (acetonitrile) and softened PMMA from filling the channels. Once the sealing step is complete, the sacrificial layer is melted and removed, leaving enclosed microfluidic channels. We found that PMMA substrates welded together using this method could withstand internal pressures of >2250 psi, more than 1 order of magnitude higher than their thermally bonded counterparts. To demonstrate the usefulness of this method, microchip capillary electrophoresis (CE) devices in PMMA were created and tested. Amino acid and peptide mixtures were separated in <15 s, with >40,000 theoretical plates in a 2.5-cm separation distance. Electric fields as high as 1.5 kV/cm were applied in these microchips, and >300 CE runs were performed on a single device with no degradation of separation performance. The simplicity of the methods presented here and the improved robustness of the resulting devices should facilitate the broader implementation of polymer microchips in microfluidic analyses.     

Gradient elution moving boundary electrophoresis for high-throughput multiplexed microfluidic devices

A novel method for performing electrophoretic separations is described-gradient elution moving boundary electrophoresis (GEMBE). The technique utilizes the electrophoretic migration of chemical species in combination with variable hydrodynamic bulk counterflow of the solution through a separation capillary or microfluidic channel. Continuous sample introduction is used, eliminating the need for a sample injection mechanism. Only analytes with an electrophoretic velocity greater than the counterflow velocity enter the separation channel. The counterflow velocity is varied over time so that each analyte is brought into the separation column at different times, allowing for high-resolution separations in very short channels. The new variable of bulk flow acceleration affords a new selectivity parameter to electrophoresis analogous to gradient elution compositions in chromatography. Because it does not require extra channels or access ports to form an injection zone and because separations can be performed in very short channels, GEMBE separations can be implemented in much smaller areas on a micro-fluidic chip as compared to conventional capillary electrophoresis. Demonstrations of GEMBE separations of small dye molecules, amino acids, DNA, and immunoassay products are presented. A low-cost, polymeric, eight-channel multiplexed microfluidic device was fabricated to demonstrate the reduced area requirements of GEMBE; the device was less than 1 in.2 in area and required only n + 1 fluidic access ports per n analyses (in this instance, nine ports for eight analyses). Parallel separations of fluorescein and carboxyfluorescein yielded less than 3% relative standard deviation (RSD) in interchannel migration times and less than 5% RSD in both peak and height measurements. The device was also used to generate a calibration curve for a homogeneous insulin immunoassay using each of the eight channels as a calibration point with less than 5% RSD at each point with replicate analyses.     

An electrospray ionization source for integration with microfluidics

We have demonstrated a new electrospray ionization (ESI) device incorporating a tip made from a shaped thin film, bonded to a microfluidic channel, and interfaced to a time-of-flight mass spectrometer (TOFMS). A triangular-shaped thin polymer tip was formed by lithography and etching. A microfluidic channel, 20 microm wide and 10 microm deep, was embossed in a cyclo olefin substrate using a silicon master. The triangular tip was aligned with the channel and bonded between the channel plate and a flat plate to create a microfluidic channel with a wicking tip protruding from the end. This structure aided the formation of a stable Taylor cone at the apex of the tip, forming an electrospray ionization source. This source was tested by spraying several solutions for mass spectrometric analysis. Because the components are all made by lithographic approaches with high geometrical fidelity, an integrated array system with multiple channels can be formed with the same method and ease as a single channel. We tested a multichannel system in a multiplexed manner and showed reliable operation with no significant cross contamination between closely spaced channels.     

Anomalous radial migration of single DNA molecules in capillary electrophoresis

We report the unexpected radial migration of DNA molecules in capillary electrophoresis (CE) with applied Poiseuille flow. Such movement can contribute to anomalous migration times, peak dispersion, and size and shape selectivity in CE. When Poiseuille flow is applied from the cathode to the anode, DNA molecules move toward the center of the capillary, forming a narrow, highly concentrated zone. Conversely, when the flow is applied from the anode to the cathode, DNA molecules move toward the walls, leaving a DNA-depleted zone around the axis. We showed that the deformation and orientation of DNA molecules under Poiseuille flow was responsible for the radial migration. By analyzing the forces acting on the deformed and oriented DNA molecules, we derived an expression for the radial lift force, which explained our results very well under different conditions with Poiseuille flow only, electrophoresis only, and the combination of Poiseuille flow and electrophoresis. Factors governing the direction and velocity of radial migration were elucidated. Potential applications of this phenomenon include an alternative to sheath flow in flow cytometry, improving precision and reliability of single-molecule detection, reduction of wall adsorption, and size separation with a mechanism akin to field-flow fractionation. On the negative side, nonuniform electroosmotic flow along the capillary or microfluidic channel is common in CE, and radial migration of certain analytes cannot be neglected.     

Capillary electrophoresis separation in the presence of an immiscible boundary for droplet analysis

This paper demonstrates the ability to use capillary electrophoresis (CE) separation coupled with laser-induced fluorescence for analyzing the contents of single femtoliter-volume aqueous droplets. A single droplet was formed using a T-channel (3 microm wide by 3 microm tall) connected to microinjectors, and then the droplet was fluidically moved to an immiscible boundary that isolates the CE channel (50 microm wide by 50 microm tall) from the droplet generation region. Fusion of the aqueous droplet with the immiscible boundary effectively injects the droplet content into the separation channel. In addition to injecting the contents of droplets, we found aqueous samples can be introduced directly into the separation channel by reversibly penetrating and resealing the immiscible partition. Because droplet generation in channels requires hydrophobic surfaces, we have also investigated the advantages to using all hydrophobic channels versus channel systems with patterned hydrophobic and hydrophilic regions. To fabricate devices with patterned surface chemistry, we have developed a simple strategy based on differential wetting to deposit selectively a hydrophilic polymer (poly(styrenesulfonate)) onto desired regions of the microfluidic chip. Finally, we applied our device to the separation of a simple mixture of fluorescein-labeled amino acids contained within a approximately 10-fL droplet.     

Surface modification of glycidyl-containing poly(methyl methacrylate) microchips using surface-initiated atom-transfer radical polymerization

Fabrication of microfluidic systems from polymeric materials is attractive because of simplicity and low cost. Unfortunately, the surfaces of many polymeric materials can adsorb biological samples. Therefore, it is necessary to modify their surfaces before these polymeric materials can be used for separation and analysis. Oftentimes it is difficult to modify polymeric surfaces because of their resistance to chemical reaction. Recently, we introduced a surface-reactive acrylic polymer, poly(glycidyl methacrylate-co-methyl methacrylate) (PGMAMMA), which can be modified easily and is suitable for fabrication of microfluidic devices. Epoxy groups on the surface can be activated by air plasma treatment, hydrolysis, or aminolysis. In this work, the resulting hydroxyl or amino groups were reacted with 2-bromoisobutylryl bromide to introduce an initiator for surface-initiated atom-transfer radical polymerization (SI-ATRP). Polyethylene glycol (PEG) layers grown on the surface using this method were uniform, hydrophilic, stable, and resistant to protein adsorption. Contact angle measurement and X-ray photoelectron spectroscopy (XPS) were used to characterize activated polymer surfaces, initiator-bound surfaces, and PEG-grafted surfaces. We obtained excellent capillary electrophoresis (CE) separations of proteins and peptides with the PEG-modified microchips. A separation efficiency of 4.4 x 10(4) plates for a 3.5 cm long separation channel was obtained.     

Fully packed capillary electrochromatographic microchip with self-assembly colloidal silica beads

A fully packed capillary electrochromatographic (CEC) microchip showing improved solution and chip handling was developed. Microchannels for the CEC microchip were patterned on a cyclic olefin copolymer substrate by injection molding and packed fully with 0.8-microm monodisperse colloidal silica beads utilizing a self-assembly packing technique. The silica packed chip substrate was covered and thermally press-bonded. After fabrication, the chip was filled with buffer solution by self-priming capillary action. The self-assembly packing at each channel served as a built-in nanofilter allowing quick loading of samples and running buffer solution without filtration. Because of a large surface area-to-volume ratio of the silica packing, reproducible control of electroosmotic flow was possible without leveling of the solutions in the reservoirs resulting 1.3% rsd in migration rate. The capillary electrophoretic separation characteristics of the chip were studied using fluorescein isothiocyanate (FITC)-derivatized amino acids as probe molecules. A mixture of FITC and four FITC-derivatized amino acids was successfully separated with 2-mm separation channel length.     

Microfabricated system for parallel single-cell capillary electrophoresis

Performing single-cell electrophoresis separations using multiple parallel microchannels offers the possibility of both increasing throughput and eliminating cross-contamination between different separations. The instrumentation for such a system requires spatial and temporal control of both single-cell selection and lysis. To address these problems, a compact platform is presented for single-cell capillary electrophoresis in parallel microchannels that combines optical tweezers for cell selection and electromechanical lysis. Calcein-labeled acute myloid leukemia (AML) cells were selected from an on-chip reservoir and transported by optical tweezers to one of four parallel microfluidic channels. Each channel entrance was manufactured by F2-laser ablation to form a 20- to 10-microm tapered lysis reservoir, creating an injector geometry effective in confining the cellular contents during mechanical shearing of the cell at the 10-microm capillary entrance. The contents of individual cells were simultaneously injected into parallel channels resulting in electrophoretic separation as recorded by laser-induced fluorescence of the labeled cellular contents.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.     

Immobilization of biomolecules on cycloolefin polymer supports

Recent trends in the development of microfluidic and biodiagnostic chips favor polymer materials over glass, primarily for optical and economical reasons. Therefore, existing chemical methods to prepare biomolecule microarrays on glass slides have to be adapted or replaced in order to suit polymer substrates. Here we present a strategy to immobilize DNA and antibodies on cyclic polyolefin slides, like Zeonor. This polymer represents a class of new polymeric materials with excellent optical and mechanical properties. By plasma and liquid chemical treatment followed by coating with polyelectrolytes, we have succeeded in immobilizing DNA onto the polymer substrate, yielding stable and versatile biosensor surfaces. We demonstrate the stability and usage of the coated Zeonor substrates not only in DNA chip technology but also in protein chip technology with DNA-directed immobilization of proteins.     

A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis/electrospray mass spectrometry

A novel microfabricated device was implemented for facile coupling of capillary electrophoresis with mass spectrometry (CE/MS). The device was constructed from glass wafers using standard photolithographic/wet chemical etching methods. The design integrated (a) sample inlet ports, (b) the separation channel, (c) a liquid junction, and (d) a guiding channel for the insertion of the electrospray capillary, which was enclosed in a miniaturized subatmospheric electrospray chamber of an ion trap MS. The replaceable electrospray capillary was precisely aligned with the exit of the separation channel by a microfabricated guiding channel. No glue was necessary to seal the electrospray capillary. This design allowed simple and fast replacement of either the microdevice or the electrospray capillary. The performance of the device was tested for CE/MS of peptides, proteins, and protein tryptic digests. On-line tandem mass spectrometry was used for the structure identification of the protein digest products. High-efficiency/high-resolution separations could be obtained on a longer channel (11 cm on-chip) microdevice, and fast separations (under 50 s) were achieved with a short (4.5 cm on-chip) separation channel. In the experiments, both electrokinetic and pressure injections were used. The separation efficiency was comparable to that obtained from conventional capillary electrophoresis.     

侧壁四电极电容耦合非接触电导检测器(英文)

研制了一种集成于硅基电泳芯片分离沟道末端侧壁的新型四电极电容耦合非接触电导检测器.研究了该电导检测器的等效模型,对等效电路模型中的参数进行了公式推导,并讨论了影响电导检测响应灵敏度的相关因素.采用深刻蚀及离子注入加工技术制得了用于电导检测的立体电极.制作了基于锁相放大原理的信号处理电路,对该电导检测的频率响应及灵敏度进行了测试分析.实验结果表明,当激励信号频率为300 kHz时,该电导检测器具有最佳线性度;不同浓度Na+溶液响应电压差值为5 mV;检测限达到10-8mol/L;且成功实现了Na+和Li+混合无机阳离子的电泳分离在线检测.     

Chip-based quantitative capillary electrophoresis/mass spectrometry determination of drugs in human plasma

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the on-chip separation and coupled electrospray detection of selected small drug molecule compounds. These studies include the quantitative determination of carnitine and acetylcarnitine in analytical standard solutions as well as imipramine and desipramine in fortified human plasma samples. A clinical human plasma sample was also analyzed following the normal administration of desipramine to a volunteer, and the parent drug was determined using the described chipbased CE/MS technique. In each instance, stable isotope-incorporated internal standards were used. The chip-based CE system was microfabricated from glass and coupled to a micro ion spray device constructed in-house. The atmospheric pressure ionization system employed in this work was a PE Sciex API III tandem triple quadrupole system operated in the selected ion monitoring (SIM) mode. The results from the work reported here demonstrate the feasibility for carrying out rapid (30 s) chipbased quantitative CE/MS determinations of samples containing small-molecule compounds. Using SIM CE/ MS techniques, the described API III quadrupole system provided acceptable ion current electropherograms from subpicomole levels of the targeted compounds loaded onto the chip. The corresponding electropherograms for the standard solution of carnitines at the 1-500 microg/mL level were obtained via SIM CE/MS techniques (R2 > 0.99). In addition, analyses of fortified samples of imipramine desipramine were measured relative to their corresponding d3 internal standards to obtain calibration curves ranging from 5 to 500 microg/mL in human plasma (R2 > 0.99). The intra-assay precision ranged from 4.1 to 7.3% RSD. The intra-assay accuracy ranged from 94.0 to 104%. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric determination in applications for bioanalytical measurements for these important compounds in synthetic mixtures and human plasma extracts.     

Fully microfabricated and integrated SU-8-based capillary electrophoresis-electrospray ionization microchips for mass spectrometry

We present a fully microfabricated and monolithically integrated capillary electrophoresis (CE)-electrospray ionization (ESI) chip for coupling with high-throughput mass spectrometric (MS) analysis. The chips are fabricated fully of a negative photoresist SU-8 by a standard lithographic process which enables straightforward batch fabrication of multiple chips with precisely controlled dimensions and, thus, reproducible analytical performance from chip to chip. As the coaxial sheath flow interface is patterned as an integral part of the SU-8 chip, the fluidic design is dead-volume-free. No significant peak broadening occurs so that very narrow peak widths (down to 2-3 s) are obtained. The sheath flow interface also enables comprehensive optimization of both the CE and the ESI conditions separately so that the same chip design is adaptable to diverse analytical conditions. Plate numbers of the order of 105 m-1 and good resolution are routinely reached for small molecules and peptides within a 2 cm separation length and a typical cycle time of only 30-90 s per sample. In addition, a limit of detection of 100 nM corresponding to a total amount of only 4.5 amol (per injection volume of 45 pL) and excellent quantitative linearity (R2 = 0.9999; 100 nM to 100 microM) were obtained in small-molecule analysis using verapamil as a test compound. The quantitative repeatability was proven good (8.5-21.4% relative standard deviation, peak area) also for the other drug substances and peptides tested.     

High-speed free-flow electrophoresis on chip

A microfluidic device has been developed for continuous separation in free-flow electrophoresis (FFE) mode. A mixture of two fluorescent reagents is separated into two component streams in 75 ms using a sample flow rate of 2 nL/s. The residence time of sample in the whole separation compartment is 2 s. The separation bed volume is 0.2 microL. The chip has also been used for free-flow electrophoresis of fluorescein-5-isothiocyanate-labeled amino acids in both aqueous and binary media. The short residence time and small sample flow rate make the FFE chip feasible for on-line monitoring on production lines and other chemical or biochemical processes. The in-house-made chip was composed of a plain glass substrate of 1.5-mm thickness and a PDMS layer of 0.3-mm thickness with micromachined channels. The channel design presented in this paper is versatile. With the same kind of PDMS substrates, chips for various purposes can be made depending on the locations of the reservoirs, which are cut out on the PDMS substrate. The results presented verify the scaling laws and allow prediction of FFE performances comparable to what is now state of the art on capillary electrophoresis chips.     

Two-dimensional protein separation with advanced sample and buffer isolation using microfluidic valves

Methods are described to achieve more efficient multidimensional protein separation in a microfluidic channel. The new methods couple isoelectric focusing (IEF) with high ionic strength electrophoretic separations by active microvalve control in a microchip. Several experiments demonstrating independent 2D separation were performed, and critical parameters for optimal chip performance were identified, including channel passivation, electroosmosis control, and IEF linearity control. This strategy can be used for integration of different heterogeneous separation techniques, such as IEF, capillary electrophoresis, and liquid chromatography. This new device can be ideal for preseparation and preconcentration of complex biomolecule samples for a streamlined biomolecule analysis using mass spectrometry.     

Parallel electrophoretic analysis of segmented samples on chip for high-throughput determination of enzyme activities

Capillary electrophoresis (CE) on microfabricated structures has achieved impressive sample throughput by combining fast separation speed and parallel operations. One obstacle to further increasing throughput has been lack of methods for loading and injecting individual samples at a rate that matches analysis speed. To address this issue, we have developed a microfluidic device in which samples stored as nanoliter volume plugs segmented by a fluorocarbon oil are introduced sequentially to an array of three electrophoresis channels. A microfluidic interface consisting of patterned surface chemistry and geometric restriction was used to extract samples from each segmented flow channel and transfer to the respective electrophoresis channel for separation. Fluorescence detection was achieved by imaging the chip using a fluorescence microscope equipped with a charge-coupled device. Characterization of the system shows that injection volume is controlled by sample plug volume, flow rate during introduction, and voltage applied to the electrophoresis channel. The system was tested for a GTPase assay. Peak area ratios of enzyme product and internal standard had 6% relative standard deviations. Cross-contamination between peaks was 7%. Throughput of 120 samples in 10 min was achieved. Further development of the system may allow application to high-throughput applications such as drug screening.     

Dual-channel method for interference-free in-channel amperometric detection in microchip capillary electrophoresis

A novel in-channel amperometric detection method for microchip capillary electrophoresis (CE) has been developed to avoid the interference from applied potential used in the CE separation. Instead of a single separation channel as in conventional CE microchips, we use a dual-channel configuration consisting of two different parallel separation and reference channels. A working electrode (WE) and a reference electrode (RE) are placed equally at a distance 200 microm from its outlet on each channel. Running buffer flows through the reference channel. Our dual-channel CE microchips consist of a poly(dimethylsiloxane) (PDMS) upper plate and a glass lower plate to form a PDMS/glass hybrid chip. Amperometric signals are measured without any potential shift and interference from the applied CE potential, and CE separation maintains its high resolution because this in-channel configuration does not allow additional band broadening that is notorious in end-channel and off-channel configurations. The high performance of this new in-channel electrochemical detection methodology for CE has been demonstrated by analyzing a mixture of electrochemically active biomolecules: dopamine (DA), norepinephrine, and catechol. We have achieved a 0.1 pA detectability from the analysis of DA, which corresponds to a 1.8 nM concentration.