Kihwan Choi

Digital Microfluidic Magnetic Separation for Particle-Based Immunoassays

We introduce a new format for particle-based immunoassays relying on digital microfluidics (DMF) and magnetic forces to separate and resuspend antibody-coated paramagnetic particles. In DMF, fluids are electrostatically controlled as discrete droplets (picoliters to microliters) on an array of insulated electrodes. By applying appropriate sequences of potentials to these electrodes, multiple droplets can be manipulated simultaneously and various droplet operations can be achieved using the same device design. This flexibility makes DMF well-suited for applications that require complex, multistep protocols such as immunoassays. Here, we report the first particle-based immunoassay on DMF without the aid of oil carrier fluid to enable droplet movement (i.e., droplets are surrounded by air instead of oil). This new format allowed the realization of a novel on-chip particle separation and resuspension method capable of removing greater than 90% of unbound reagents in one step. Using this technique, we developed methods for noncompetitive and competitive immunoassays, using thyroid stimulating hormone (TSH) and 17β-estradiol (E2) as model analytes, respectively. We show that, compared to conventional methods, the new DMF approach reported here reduced reagent volumes and analysis time by 100-fold and 10-fold, respectively, while retaining a level of analytical performance required for clinical screening. Thus, we propose that the new technique has great potential for eventual use in a fast, low-waste, and inexpensive instrument for the quantitative analysis of proteins and small molecules in low sample volumes.

Single drop microextraction using commercial capillary electrophoresis instruments.

Single drop microextraction (SDME), a simple and efficient sample preconcentration method for capillary electrophoresis (CE), was performed using two different commercial CE instruments. With a CE instrument providing adjustable forward and backward pressures, a single drop of an aqueous acceptor phase covered with a thin layer of an organic phase was formed at the capillary tip by programming the sample-handling functions of the instrument to perform 3-phase SDME where the organic film is essentially a membrane. Analytes from an acidic donor phase were concentrated into a basic acceptor phase yielding 190-fold enrichment in 10 min. When the donor phase was agitated using a microstirrer, a 2000-fold enrichment was obtained in 10 min. For a less flexible CE instrument, 2-phase SDME was carried out with a large volume pentanol drop held by a Teflon sleeve, yielding 110-fold enrichments in 30 min. We demonstrate a practical and automated SDME-CE technique with accuracy and reproducibility that is easy to practice to attain matrix isolation and high concentration sensitivity.

Selective preconcentration of amino acids and peptides using single drop microextraction in-line coupled with capillary electrophoresis

Single drop microextraction (SDME) can be in-line coupled with capillary electrophoresis by attaching a drop to the tip of a capillary. With a 2-layer drop comprised of an aqueous basic acceptor phase covered with a thin organic layer, acidic analytes in an aqueous acidic donor phase can be extracted into the organic layer and then back-extracted into the acceptor phase. However, preconcentration of amino acids and peptides by SDME is difficult since their zwitterionic properties prevent them from being partitioned in the middle organic phase. When amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), amino acids without a charged side chain were converted to carboxylic acids. In the acidic donor phase, those NBD-amino acids were predominantly neutral and they were successfully concentrated into the basic acceptor phase. In the meantime, amino acids with a charged side chain after NBD-F derivatization were not concentrated via SDME. With this selective SDME, we were able to extract acidic and neutral amino acids obtaining several hundred-fold enrichments within 5 min at 25 degrees C, while leaving basic amino acids-Arg, Lys, and His-in the acidic donor phase. Furthermore, detection sensitivity was enhanced by employing laser-induced fluorescence detection. We then applied this technique to the selective concentration of peptides.

Near infrared dye indocyanine green doped silica nanoparticles for biological imaging

Indocyanine green (ICG) is an FDA-approved near infrared (NIR) fluorescent dye used in clinical imaging. However, its applications remain limited due to its short half-life, nonspecific plasma binding, optical instability, and poor aqueous stability. Dye doped silica nanoparticles provide an effective barrier in keeping the dye away from the surrounding environment, but ICG cannot be encapsulated into silica easily by conventional methods. In this study, ICG molecules ion-paired with a cationic polymer polyethylenimine (PEI) were successfully encapsulated into a silica matrix to form ICG doped silica nanoparticles by using the Stöber method. Pairing with PEI reduced self-quenching of fluorescence by preventing the aggregation of ICG molecules in silica nanoparticles. Dye leakage was also reduced to the level of 3-6% loss in 5 days. NIR fluorescence images of ICG doped silica NPs below a 2.0 cm thick porcine muscle sample illuminated by NIR light were obtained.

Digital Microfluidic Platform for the Detection of Rubella Infection and Immunity: A Proof of Concept

BACKGROUND Whereas disease surveillance for infectious diseases such as rubella is important, it is critical to identify pregnant women at risk of passing rubella to their offspring, which can be fatal and can result in congenital rubella syndrome (CRS). The traditional centralized model for diagnosing rubella is cost-prohibitive in resource-limited settings, representing a major obstacle to the prevention of CRS. As a step toward decentralized diagnostic systems, we developed a proof-of-concept digital microfluidic (DMF) diagnostic platform that possesses the flexibility and performance of automated immunoassay platforms used in central facilities, but with a form factor the size of a shoebox. METHODS DMF immunoassays were developed with integrated sample preparation for the detection of rubella virus (RV) IgG and IgM. The performance (sensitivity and specificity) of the assays was evaluated with serum and plasma samples from a commercial antirubella mixed-titer performance panel. RESULTS The new platform performed the essential processing steps, including sample aliquoting for 4 parallel assays, sample dilution, and IgG blocking. Testing of performance panel samples yielded diagnostic sensitivity and specificity of 100% and 100% for both RV IgG and RV IgM. With 1.8 μL sample per assay, 4 parallel assays were performed in approximately 30 min with <10% mean CV. CONCLUSIONS This proof of concept establishes DMF-powered immunoassays as being potentially useful for the diagnosis of infectious disease.

In-line coupling of two-phase single drop microextraction and large volume stacking using an electroosmotic flow pump in nonaqueous capillary electrophoresis

In order to improve the concentration sensitivity of capillary electrophoresis (CE), two sample preconcentration techniques, single drop microextraction (SDME) and large volume stacking using an electroosmotic flow pump (LVSEP), were coupled in-line in a commercial CE instrument. By simple programming of liquid handling sequences, a pentanol drop was prepared at the tip of a fused silica capillary over which a Teflon tube had been sleeved to serve as a hydrophobic support. After extraction of the analytes from an aqueous donor solution into the drop, the entire capillary column was filled with enriched pentanol extract. LVSEP, in which the sample matrix is automatically removed by the EOF, was then carried out using a methanolic run buffer. The overall enrichment factors for the analytes pentachlorophenol (PCP), 3-bromobenzoic acid (3-BBA), and 4-iodobenzoic acid (4-IBA), from a combination of 30min SDME and LVSEP on a 27cm capillary, were about 7000, even without agitation of the donor solution. The resulting limits of detection for PCP, 3-BBA, and 4-IBA were 0.7, 0.3 and 0.7nM, respectively. Since no modification of the existing CE instrument is necessary and a bare capillary is used for LVSEP, this scheme can be adapted quite easily for many CE applications that require high concentration sensitivity.

Direct chiral analysis of primary amine drugs in human urine by single drop microextraction in‐line coupled to CE

Three‐phase single drop microextraction (SDME) was in‐line coupled to chiral CE of weakly basic amine compounds including amphetamine. SDME was used for the matrix isolation and sample preconcentration in order to directly analyze urine samples with the minimal pretreatment of adding NaOH. A small drop of an acidic aqueous acceptor phase covered with a thin layer of octanol was formed at the tip of a capillary by simple manipulation of the liquid handling functions of a commercial CE instrument. While the saline matrix of the urine sample was blocked by the octanol layer, the basic analytes in a basic aqueous donor phase were concentrated into the acidic acceptor drop through the octanol layer by the driving force of the pH difference between the two aqueous phases. The enantiomers of the enriched amines were resolved by using (+)‐(18‐crown‐6)‐tetracarboxylic acid as a chiral selector for the subsequent CE separation. From 10 min SDME with the agitation of the donor phase by a small stirrer retrofit to the CE instrument, enrichment factors were about a 1000‐fold, yielding the LOD of 0.5 ng/mL for amphetamine. This low LOD value as well as the convenience of in‐line coupled SDME make the proposed scheme well suited for the demanding chiral analysis of amphetamine‐type stimulants.

Highly sensitive chiral analysis of amino acids by in-line single drop microextraction and capillary electrophoresis with laser-induced fluorescence detection.

A highly sensitive method for chiral analysis of amino acids by in-line single drop microextraction (SDME) and chiral capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was developed. In SDME, a drop of a basic aqueous acceptor phase covered with a thin organic layer was formed at the tip of a capillary by simple combination of sample-handling sequences of a CE apparatus. Then fluorescein isothiocyanate (FITC)-derivatized amino acids in an acidic donor solution were enriched into the drop through the organic layer. The enriched enantiomers were then resolved using a dual chiral selector of β-cyclodextrin (β-CD) and sodium taurodeoxycholate (STC). Here, in addition to serving as a labeling reagent providing high fluorescence signal, hydrophobic FITC was primarily used as a modifier aiding the extraction of zwitterionic amino acids by blocking the amino groups and increasing the hydrophobicity, yielding 220 times increase in extraction efficiency. Several hundred-fold enrichments were achieved with 10 min SDME, yielding LODs of 30-60 pM and enabling direct analysis of d-AAs in a 99% enantiomeric excess mixture. In view of no additional modification of the existing commercial CE instrument, this method without stirring can be easily realized using known operations. When a microstirrer was customized to the CE instrument several thousand-fold enrichments could be obtained with LODs in the low picomolar range of 1-3 pM.

Electrochemiluminescence on digital microfluidics for microRNA analysis.

Electrochemiluminescence (ECL) is a sensitive analytical technique with great promise for biological applications, especially when combined with microfluidics. Here, we report the first integration of ECL with digital microfluidics (DMF). ECL detectors were fabricated into the ITO-coated top plates of DMF devices, allowing for the generation of light from electrically excited luminophores in sample droplets. The new system was characterized by making electrochemical and ECL measurements of soluble mixtures of tris(phenanthroline)ruthenium(II) and tripropylamine (TPA) solutions. The system was then validated by application to an oligonucleotide hybridization assay, using magnetic particles bearing 21-mer, deoxyribose analogues of the complement to microRNA-143 (miRNA-143). The system detects single nucleotide mismatches with high specificity, and has a limit of detection of 1.5 femtomoles. The system is capable of detecting miRNA-143 in cancer cell lysates, allowing for the discrimination between the MCF-7 (less aggressive) and MDA-MB-231 (more aggressive) cell lines. We propose that DMF-ECL represents a valuable new tool in the microfluidics toolbox for a wide variety of applications.

Sensitive analysis of amino acids with carrier-mediated single drop microextraction in-line coupled with capillary electrophoresis

In order to analyze amino acids sensitively without derivatization, we have developed carrier-mediated single drop microextraction (SDME). Nonane-1-sulfonic acid was added to an acidic sample donor solution as a carrier to form neutral ion pair complexes with amino acids. The ion pair complexes were extracted to the organic phase, covering a drop of an aqueous basic acceptor phase hanging at the tip of a capillary, and then back-extracted to the basic acceptor phase, where both the amino acids and the carrier have negative charges and the ion pair complexes are broken. The resulting extract of enriched amino acids was injected into the capillary and analyzed by capillary electrophoresis. With 20-min SDME with agitation of the donor phase, enrichment factors of four aromatic amino acids were up to 120-fold, yielding the LOD of 70-500 nM. The linear dynamic ranges for corrected peak areas were 1-100 μM with linear correlation coefficients larger than 0.9959. With internal standardization, the intraday RSDs of migration times and corrected peak areas were 0.01-0.04% and 2.0-3.7%, respectively. The capabilities of sample cleanup including desalting and preconcentration of carrier-mediated SDME were demonstrated with the analysis of human urine after minimal pretreatment of acidification and centrifugation.