Towards clinically translatable in vivo nanodiagnostics
Nature Reviews Materials
View details for DOI 10.1038/natrevmats.2017.14
Molecular profiling of single circulating tumor cells from lung cancer patients
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (52): E8379-E8386
Circulating tumor cells (CTCs) are established cancer biomarkers for the "liquid biopsy" of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non-small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.
View details for DOI 10.1073/pnas.1608461113
View details for Web of Science ID 000391090800003
View details for PubMedID 27956614
View details for PubMedCentralID PMC5206556
A method for nanofluidic device prototyping using elastomeric collapse
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (37): 15549-15554
Nanofluidics represents a promising solution to problems in fields ranging from biomolecular analysis to optical property tuning. Recently a number of simple nanofluidic fabrication techniques have been introduced that exploit the deformability of elastomeric materials like polydimethylsiloxane (PDMS). These techniques are limited by the complexity of the devices that can be fabricated, which can only create straight or irregular channels normal to the direction of an applied strain. Here, we report a technique for nanofluidic fabrication based on the controlled collapse of microchannel structures. As is demonstrated, this method converts the easy to control vertical dimension of a PDMS mold to the lateral dimension of a nanochannel. We demonstrate here the creation of complex nanochannel structures as small as 60 nm and provide simple design rules for determining the conditions under which nanochannel formation will occur. The applicability of the technique to biomolecular analysis is demonstrated by showing DNA elongation in a nanochannel and a technique for optofluidic surface enhanced Raman detection of nucleic acids.
View details for DOI 10.1073/pnas.0904004106
View details for Web of Science ID 000269806600009
View details for PubMedID 19717418
High-Density Lipoprotein Nanoparticle Imaging in Atherosclerotic Vascular Disease
JACC: Basic to Translational Science
2017; 2 (1): 98-100
View details for DOI 10.1016/j.jacbts.2017.01.005
High-throughput full-length single-cell mRNA-seq of rare cells.
2017; 12 (11): e0188510
Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.
View details for DOI 10.1371/journal.pone.0188510
View details for PubMedID 29186152
View details for PubMedCentralID PMC5706670
Capture and Genetic Analysis of Circulating Tumor Cells Using a Magnetic Separation Device (Magnetic Sifter).
Methods in molecular biology (Clifton, N.J.)
2017; 1634: 153?62
Circulating tumor cells (CTCs) are currently widely studied for their potential application as part of a liquid biopsy. These cells are shed from the primary tumor into the circulation, and are postulated to provide insight into the molecular makeup of the actual tumor in a minimally invasive manner. However, they are extremely rare in blood, with typical concentrations of 1-100 in a milliliter of blood; hence, a need exists for a rapid and high-purity method for isolating CTCs from whole blood. Here, we describe the application of a microfabricated magnetic sifter toward isolation of CTCs from whole blood at volumetric flow rates of 10 mL/h, along with the use of a PDMS-based nanowell system for single-cell gene expression profiling. This method allows rapid isolation of CTCs and subsequent integration with downstream genetic profiling methods for clinical applications such as targeted therapy, therapy monitoring, or further biological studies.
View details for DOI 10.1007/978-1-4939-7144-2_12
View details for PubMedID 28819848
Multigene Profiling of Single Circulating Tumor Cells
Molecular & Cellular Oncology
2017; 4 (2): e1289295
Numerous techniques for isolating circulating tumor cells (CTCs) have been developed. Concurrently, single-cell techniques that can reveal molecular components of CTCs have become widely available. We discuss how the combination of isolation and multigene profiling of single CTCs in our platform can facilitate eventual translation to the clinic.
View details for DOI 10.1080/23723556.2017.1289295
View details for PubMedCentralID PMC5383366
Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA.
Cell-free DNA (cfDNA) diagnostics are emerging as a new paradigm of disease monitoring and therapy management. The clinical utility of these diagnostics is relatively limited by a low signal-to-noise ratio, such as with low allele frequency (AF) mutations in cancer. While enriching for rare alleles to increase their AF before sample analysis is one strategy that can greatly improve detection capability, current methods are limited in their generalizability, ease of use, and applicability to point mutations.Leveraging the robust single-base-pair specificity and generalizability of the CRISPR associated protein 9 (Cas9) system, we developed a deactivated Cas9 (dCas9)-based method of minor-allele enrichment capable of efficient single-target and multiplexed enrichment. The dCas9 protein was complexed with single guide RNAs targeted to mutations of interest and incubated with cfDNA samples containing mutant strands at low abundance. Mutation-bound dCas9 complexes were isolated, dissociated, and the captured DNA purified for downstream use.Targeting the 3 most common epidermal growth factor receptor mutations (exon 19 deletion, T790M, L858R) found in nonsmall-cell lung cancer (NSCLC), we achieved >20-fold increases in AF and detected mutations by use of qPCR at an AF of 0.1%. In a cohort of 18 NSCLC patient-derived cfDNA samples, our method enabled detection of 8 out of 13 mutations that were otherwise undetected by qPCR.The dCas9 method provides important application of the CRISPR/Cas9 system outside the realm of genome editing and can provide a step forward for the detection capability of cfDNA diagnostics.
View details for DOI 10.1373/clinchem.2017.278911
View details for PubMedID 29038154
Dual transcript and protein quantification in a massive single cell array.
Lab on a chip
2016; 16 (19): 3682-3688
Recently, single-cell molecular analysis has been leveraged to achieve unprecedented levels of biological investigation. However, a lack of simple, high-throughput single-cell methods has hindered in-depth population-wide studies with single-cell resolution. We report a microwell-based cytometric method for simultaneous measurements of gene and protein expression dynamics in thousands of single cells. We quantified the regulatory effects of transcriptional and translational inhibitors on cMET mRNA and cMET protein in cell populations. We studied the dynamic responses of individual cells to drug treatments, by measuring cMET overexpression levels in individual non-small cell lung cancer (NSCLC) cells with induced drug resistance. Across NSCLC cell lines with a given protein expression, distinct patterns of transcript-protein correlation emerged. We believe this platform is applicable for interrogating the dynamics of gene expression, protein expression, and translational kinetics at the single-cell level - a paradigm shift in life science and medicine toward discovering vital cell regulatory mechanisms.
View details for DOI 10.1039/c6lc00762g
View details for PubMedID 27546183
View details for PubMedCentralID PMC5221609
Pref-1 Marks Very Early Mesenchymal Precursors Required for Adipose Tissue Development and Expansion
2014; 8 (3): 678-687
Pref-1 is an EGF-repeat-containing protein that inhibits adipocyte differentiation. To better understand the origin and development of white adipose tissue (WAT), we generated transgenic mouse models for transient or permanent fluorescent labeling of cells using the Pref-1 promoter, facilitating inducible ablation. We show that Pref-1-marked cells retain proliferative capacity and are very early adipose precursors, prior to expression of Zfp423 or PPAR?. In addition, the Pref-1-marked cells establish that adipose precursors are mesenchymal, but not endothelial or pericytal, in origin. During embryogenesis, Pref-1-marked cells first appear in the dorsal mesenteric region as early as embryonic day 10.5 (E10.5). These cells become lipid-laden adipocytes at E17.5 in the subcutaneous region, whereas visceral WAT develops after birth. Finally, ablation of Pref-1-marked cells prevents not only embryonic WAT development but also later adult adipose expansion upon high-fat feeding, demonstrating the requirement of Pref-1 cells for adipogenesis.
View details for DOI 10.1016/j.celrep.2014.06.060
View details for Web of Science ID 000341572200005
View details for PubMedID 25088414
View details for PubMedCentralID PMC4138044
Toward Integrated Molecular Diagnostic System (iMDx): Principles and Applications
IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING
2014; 61 (5): 1506-1521
Integrated molecular diagnostic systems ( iMDx), which are automated, sensitive, specific, user-friendly, robust, rapid, easy-to-use, and portable, can revolutionize future medicine. This review will first focus on the components of sample extraction, preservation, and filtration necessary for all point-of-care devices to include for practical use. Subsequently, we will look for low-powered and precise methods for both sample amplification and signal transduction, going in-depth to the details behind their principles. The final field of total device integration and its application to the clinical field will also be addressed to discuss the practicality for future patient care. We envision that microfluidic systems hold the potential to breakthrough the number of problems brought into the field of medical diagnosis today.
View details for DOI 10.1109/TBME.2014.2309119
View details for Web of Science ID 000335150300015
View details for PubMedID 24759281
View details for PubMedCentralID PMC4141683
Hemolysis-free blood plasma separation
LAB ON A CHIP
2014; 14 (13): 2287-2292
Hemolysis, involving the rupture of red blood cells (RBCs) and release of their contents into blood plasma, is a major issue of concern in clinical fields. Hemolysis in vitro can occur as a result of errors in clinical trials; in vivo, hemolysis can be caused by a variety of medical conditions. Blood plasma separation is often the first step in blood-based clinical diagnostic procedures. However, inhibitors released from RBCs due to hemolysis during plasma separation can lead to problems in diagnostic tests such as low sensitivity, selectivity and inaccurate results. In particular, a general lack of simple and reliable blood plasma separation methods has been a major obstacle for microfluidic-based point-of-care (POC) diagnostic devices. Here we present a hemolysis-free microfluidic blood plasma separation platform. A membrane filter was positioned on top of a vertical up-flow channel (filter-in-top configuration) to reduce clogging of RBCs by gravity-assisted cells sedimentation. With this device, separated plasma volume was increased approximately 4-fold (2.4 ?L plasma after 20 min with 38% hematocrit human whole blood), and hemoglobin concentration in separated plasma was decreased approximately 90% due to the prevention of RBCs hemolysis, when compared to conventional filter-in-bottom configuration blood plasma separation platforms. On-chip plasma contained ~90% of protein and ~100% of nucleic acids found in off-chip centrifuged plasma, confirming comparable target molecule recovery efficiency. This simple and robust on-chip blood plasma separation device integrates with downstream detection modules to ultimately create sample-to-answer microfluidic POC diagnostics devices.
View details for DOI 10.1039/c4lc00149d
View details for Web of Science ID 000337096800017
View details for PubMedID 24825250
Optical Methods in Studies of Olfactory System
Springer. 2014: 191?220
View details for DOI 10.1007/978-94-017-8613-3_11
- Discriminating cellular heterogeneity using microwell-based RNA cytometry. Nature communications 2014; 5: 3451-?
Rapid Prototyping of Nanofluidic Systems Using Size-Reduced Electrospun Nanofibers for Biomolecular Analysis
2010; 6 (21): 2420-2426
Biomolecular transport in nanofluidic confinement offers various means to investigate the behavior of biomolecules in their native aqueous environments, and to develop tools for diverse single-molecule manipulations. Recently, a number of simple nanofluidic fabrication techniques has been demonstrated that utilize electrospun nanofibers as a backbone structure. These techniques are limited by the arbitrary dimension of the resulting nanochannels due to the random nature of electrospinning. Here, a new method for fabricating nanofluidic systems from size-reduced electrospun nanofibers is reported and demonstrated. As it is demonstrated, this method uses the scanned electrospinning technique for generation of oriented sacrificial nanofibers and exposes these nanofibers to harsh, but isotropic etching/heating environments to reduce their cross-sectional dimension. The creation of various nanofluidic systems as small as 20 nm is demonstrated, and practical examples of single biomolecular handling, such as DNA elongation in nanochannels and fluorescence correlation spectroscopic analysis of biomolecules passing through nanochannels, are provided.
View details for DOI 10.1002/smll.201000884
View details for Web of Science ID 000284016700012
View details for PubMedID 20878634
Selection and elution of aptamers using nanoporous sol-gel arrays with integrated microheaters
LAB ON A CHIP
2009; 9 (9): 1206-1212
RNA and DNA aptamers that bind to target molecules with high specificity and affinity have been a focus of diagnostics and therapeutic research. These aptamers are obtained by SELEX (Systematic Evolution of Ligands by EXponential enrichment) often requiring more than 10 successive cycles of selection and amplification, where each cycle normally takes 2 days per cycle of SELEX. Here, we have demonstrated the use of sol-gel arrays of proteins in a microfluidic system for efficient selection of RNA aptamers against multiple target molecules. The microfluidic chip incorporates five sol-gel binding droplets, within which specific target proteins are imbedded. The droplets are patterned on top of individually addressable electrical microheaters used for selective elution of aptamers bound to target proteins in the sol-gel droplets. We demonstrate that specific aptamers bind their respective protein targets and can be selectively eluted by micro-heating. Finally, our microfluidic SELEX system greatly improved selection efficiency, reducing the number of selection cycles needed to produce high affinity aptamers. The process is readily scalable to larger arrays of sol-gel-embedded proteins. To our knowledge, this is the first demonstration of a chip-based selection of aptamers using microfluidics, thereby allowing development of a high throughput and efficient SELEX procedures.
View details for DOI 10.1039/b814993c
View details for Web of Science ID 000265223200008
View details for PubMedID 19370238
On-chip coupling of electrochemical pumps and an SU-8 tip for electrospray ionization mass spectrometry
2008; 10 (6): 891-897
We present the integration of a cyclo olefin copolymer microfluidic chip with electrochemical pumps and an SU-8 tip for electrospray ionization mass spectrometry. The electrochemical pump, using electrolysis as an internal pressure source, was fabricated directly on the surface of the polymer chip. The triangular SU-8 emitter tip was fabricated on a glass wafer using standard photolithography. After release from the glass wafer, this tip was aligned to the microchannel and bonded between two polymer plates. The electrochemical pump and the electrospray tip were tested with electrospray ionization mass spectrometry. Mass spectrometry confirmed the stability of the electrochemical pump and the electrospray tip.
View details for DOI 10.1007/s10544-008-9203-6
View details for Web of Science ID 000259246400013
View details for PubMedID 18563570
- Microfluidic encapsulated nanoelectromechanical resonators JOURNAL OF VACUUM SCIENCE & TECHNOLOGY B 2007; 25 (4): 1171-1174