Chemistries for Patterning Robust DNA MicroBarcodes Enable Multiplex Assays of Cytoplasm Proteins from Single Cancer Cells

Abstract

The optimization of chemistries to enable the patterning of miniaturized DNA barcodes using microfluidics flow channels is described (see picture). Experiment and theory reveal that solvent mixtures in which counterions are strongly associated with the negatively charged DNA oligomers may be harnessed to produce high quality, high density DNA microarray patterns over a large area. The demand for parallel, multiplex analysis of protein biomarkers from ever smaller biospecimens is an increasing trend for both fundamental biology and clinical diagnostics.1–3 The most highly multiplex protein assays rely on spatially encoded antibody microarrays,4–6 and small biospecimens samples are now routinely manipulated using microfluidics approaches. The integration of antibody microarray techniques with microfluidics chips has only been explored relatively recently. One challenge arises from the relative instability of antibodies to microfluidics fabrication conditions. In recent years, several groups have devised methods to transform standard DNA microarrays in situ into protein microarrays and cell-capture platforms.7–13 These approaches capitalize on the chemical robustness of DNA oligomers, and the reliable assembly of DNA-labeled structures via complementary hybridization. Recently, Fan et al. utilized a microfluidics-based flow patterning technique to generate DNA barcode-type arrays at 10× higher density than standard, spotted microarrays.14 The DNA barcodes were converted into antibody arrays using the DNA-encoded antibody library (DEAL) technique, and then applied towards the measurement of a highly multiplex panel of proteins from a pinprick of whole blood. A second challenge involves scaling such miniaturized DNA microarrays so that a large surface area can be encoded. This problem is non-trivial, as it involves identifying chemistries for patterning 10−5 m wide, 1 m long strips of biomolecules with a uniformity that permits those patterns to be utilized in hundreds to thousands of quantitative protein assays per chip. Herein, we explore the surface chemistry associated with microfluidics-based flow patterning of DNA barcodes, with an eye towards producing highly reproducible and robust barcodes. We then apply the optimized chemistry towards assaying a panel of cytoplasmic proteins from single cells. We explore three different flow patterning surface chemistries: two rely upon the electrostatic adsorption of DNA onto a poly-L-lysine (PLL) surface, and the third utilizes flow patterning of dendrimers onto aminated glass substrates, followed by covalent attachment of DNA oligomers onto the dendrimer scaffolds. For the electrostatic adsorption cases, we investigate, using both theory and experiment, the role that counterions play in flow patterning within the confined dimensions of a microfluidic channel, and we find that solvent mixtures which associate counterions more strongly to the negatively charged DNA oligomers yield more reproducible and robust barcodes. We then demonstrate the utility of the best flow patterning chemistry by combining it with DEAL to construct antibody barcodes for quantitatively assaying a panel of phosphorylated proteins, associated with oncogenic pathways, from single cells that are representative of the brain cancer glioblastoma multiforme (GBM). The microfluidics flow patterning chip is comprised of a patterned polydimethylsiloxane (PDMS) layer adhered to an aminated or PLL-coated glass substrate that provides the base surface for the microchannels. The microchannels are long (about 55 cm), meandering channels that span ca. 0.85 cm2 of our substrate, and are used to pattern a DNA barcode over most of the glass surface (Figure 1 b). After the flow patterning is completed, the PDMS layer is replaced with a second micropatterned PDMS layer that is designed to support a biological assay, such as the previously reported blood proteomics chip,14 or the single-cell proteomics chips utilized herein. For the microfluidic patterning method to be useful, it must generate a DNA barcode that exhibits high and uniform DNA loading over the entire substrate. We evaluated the patterning chemistries illustrated in Figure 1 a, Schemes 1–3. Schemes 1 and 2 are drawn from the conventional protocol for pin-spotted microarrays—a solution containing the DNA is introduced, the solvent is evaporated, and subsequent thermal or UV treatment is employed to cross-link the deposited DNA to the substrate. In Scheme 1 ssDNA oligomers dissolved in phosphate-buffered saline (PBS) are utilized, whereas in Scheme 2 ssDNAs in a 1:1 mixture of 1×PBS and dimethyl sulfoxide (DMSO) are employed. DMSO is used in conventional microarray preparation to improve feature consistency by reducing the rate of solvent evaporation and by denaturing the DNA15 although, as described below, its role in this process is different. In Scheme 3 a covalent immobilization method based upon a dendrimer scaffold is utilized.16 Poly(amidoamine) (PAMAM) dendrimers (generation 4.5, carboxylate surface) have previously shown promise as DNA and protein microarray substrates. Dendrimers do not form entangled chains17 and because harsh crosslinking procedures are avoided, dendrimer-immobilized DNA retains high accessibility and activity in microarray applications. Moreover, the highly branched structure of the dendrimers provides a high density of reactive sites for surface attachment and for DNA coupling, thus leading to a high overall binding capacity. For all cases, a high level of DNA loading has been shown to decrease non-specific binding when compared to standard microarray substrates.11, 18–20 a) Surface treatment schemes. b) Design of the DNA patterning device (top) and fluorescence image of DNAs filled into the channel (still in solution). Outer five channels are filled with DNAs in 1:1 mixture of PBS and water (Scheme 1 ). The five inner are filled with DNA in a 1:1 mixture of PBS and DMSO (Scheme 2 ). Three channels in between are left empty for visualization. c) Fluorescence images of patterned DNAs by three schemes. Figure 1 b (top) shows the PDMS chip design used for barcode patterning. Thirteen discrete channels (for a thirteen-element barcode) allow for a multiplex microarray. We loaded five adjacent channels according to Scheme 1, skipped three channels, and then loaded the remaining five channels according to Scheme 2. The use of fluorescently-tagged DNA permitted measurements of the DNA distribution within each individual channel immediately after introducing the solutions. Figure 1 b demonstrates a clear difference in aqueous DNA distribution across the chip: DNA loaded according to Scheme 1 (outer five channels) is notably lower in concentration near the middle of the chip (Figure 1 b, Region 2) and is barely detectable near the channel exit (Figure 1 b, Region 1). Conversely, DNA loaded according to Scheme 2 (inner five channels) presents an even, consistent distribution across the entire chip. Notably, Scheme 1 yields a relatively higher fluorescence intensity at the input side of the chip. These results clearly indicate that, for Scheme 1, the ssDNA oligomers are accumulating upstream during the early stages of flow, and so are depleted from the advancing solution by the time it reaches mid-chip. The actual patterning of the glass substrate occurs when solvent is evaporated (Figure S2, Supportiing Information). Indeed, the final patterning results after solvent evaporation and cross-linking (Figure 1 c, top) reflect the trend established by the aqueous fluorescence images; Scheme 2 produces uniform DNA barcodes across the substrate, while Scheme 1 does not. Line profiles corresponding to Figure 1 c can be found in Figure S1 (Supporting Information). In order to understand the difference in patterning uniformity between Schemes 1 and 2, we considered the electrostatic environment for each case. As depicted in Figure 2 a, the PDMS side walls carry a slightly negative zeta potential, whereas the PLL surface has a strong positive zeta potential.21 When the ssDNA solution in Scheme 1 is introduced to the channel, ssDNA near the PLL matrix is electrostatically immobilized, thereby generating a concentration gradient.22 As the solution flows towards the channel exit, the ssDNA oligomers are continually depleted via deposition onto the PLL surface. Figure 2 b shows the results from a rough simulation designed to capture the mean concentration of aqueous ssDNA as the solution traverses a channel. The simulation implies that the effect of electrostatic adsorption proves dominant even at high DNA concentrations, a result that agrees well with the observed behavior for Scheme 1 in Figure 1 b. A detailed description of the model and assumptions employed can be found in the Supporting Information. We tested this model via the strong negative charging of all four channel surfaces via O2 plasma treatment. Consistent with the model, both Schemes 1 and 2 exhibited equivalently uniform distribution of fluorescence intensity across the chip (Figure S3 b, Supporting Information). We note that lack of the positive charges on the bottom surface failed to hold DNAs during the drying procedure and that the plasma treatment induces the irreversible bonding of PDMS and glass, which limits further use beyond this experimental test. Electrostatic adsorption of DNAs on PLL surface and DMSO effect. a) The filling step. b) Simulation result of electrostatic adsorption of DNAs to PLL surface. c) Molecular simulation of the DMSO effect: the radial distribution function of P atom of the phosphate group and the sodium ions. The presence of DMSO pumps sodium ions from the 2nd shell to the 1st shell (arrow). d) Schematics for DMSO effect. Green circles represent sodium ions. The results from Schemes 1 and 2 imply that DMSO alleviates the electrostatic adsorption effect. In order to understand this more fully, we performed molecular dynamics (MD) simulations of DNA in PBS and PBS/DMSO solutions; 3 ns of NPT [NPT is a simulation in which number of moles (N), pressure (P) and temperature (T) are held constant]. The MD simulations were performed with the last 1 ns trajectory used for analysis. We examined the radial distribution function of phosphorous atoms in the DNA backbone with respect to various elements of the surrounding solvent. For example, the radial distribution function of P and the O atom of a water molecule is virtually unperturbed by the addition of DMSO (Figure S4, Supporting Information). Consequently, it is unsurprising that the radial distribution function of P and the S atom of DMSO (Figure 2 c, black solid line) reveals that DMSO is not forming a solvation structure with the DNA backbone. However, Figure 2 c demonstrates a clear interaction between P and Na+ ions, which delineates into two well-defined shell structures: the first is located at r<4.3 Å while the second is located at 4.3 Å<r<6.6 Å. These are similar to the locations of the first and the second water solvation structures. By integrating the radial distribution functions, we determined the number of molecules per phosphate in the first and second shells for both PBS and PBS/DMSO solutions. Although the number H2O molecules per shell is virtually independent of DMSO, DMSO does significantly increase the number of Na+ ions in the first shell (from 0.14 to 0.24), and it decreases the number of Na+ ions in the second shell (from 0.61 to 0.34). Conversely, the number of DMSO molecules is almost zero in the first shell (0.01) but becomes significant in the second shell (0.20). Thus, we conclude that DMSO, with a lower dielectric constant relative to water (47.2 vs 80), destabilizes the solvation energy of Na+ in the second shell. This thermodynamic change prompts the sodium ions to move to the first shell where they are stabilized by electrostatic interactions with the negatively charged phosphate groups. The increased number of sodium ions near the DNA backbone screens the negative charges of phosphate groups more efficiently, thereby reducing electrostatic interactions of the DNA with the PLL surface, resulting in uniform DNA distribution throughout the channels. Although the addition of DMSO to DNA patterning solutions yields the same ultimate effect for both traditional spotted arrays and microfluidics-patterned barcodes, the underlying mechanisms are completely different. We conclude that Scheme 2 is intrinsically superior relative to Scheme 1. We now turn towards analyzing Scheme 3, and comparing it against Scheme 2. For this scheme, the PAMAM dendrimers are first covalently attached to the aminated glass surface, and then (aminated) ssDNA oligomers are covalently attached to the dendrimers. The lack of a solvent evaporation step makes Scheme 3 significantly more rapid than Scheme 2. We flowed activated PAMAM dendrimers, followed by aminated ssDNA, through ten microfluidic channels (Figure 1 b). Note that the aqueous DNA distribution is expected to be uniform because the substrate surface is comprised of charge-neutral N-hydroxysuccinimide (NHS)-modified carboxylates which minimize electrostatic interactions. The resulting DNA microarray was assayed for uniformity with complementary DNAs labeled with Cy3-fluorophores. Visual analysis indicates good uniformity across the chip (Figure 1 c, bottom). In order to quantify the patterning quality for all three schemes, we obtained signal intensities for each channel at sixteen locations within the patterning region and calculated the coefficient of variation (CV). The CV is defined as the standard deviation divided by the mean and expressed as a percentage. CVs for Schemes 1, 2, and 3 registered 69.8 %, 10.5 %, and 10.9 %, respectively. Thus, we conclude that Schemes 2 and 3 offer consistent DNA loading across the entire substrate. Having established that Schemes 2 and 3 produce consistent, large-scale DNA barcodes, we then extended our analysis of array consistency to protein measurements. We previously demonstrated that, when using the DEAL platform for multiplex protein sensing in microfluidics channels, the sensitivities of the assays directly correlate with the amount of immobilized DNA,14 up to the point where the DNA coverage is We performed protein assays the of our DNA to that the results described into and barcodes for protein protein assays were performed in microfluidic channels which were to the patterned barcodes channels for Scheme 2 and four channels for Scheme This to microarray with a single small For barcodes using Scheme 2, we utilized the DEAL technique to into antibody barcodes designed to the phosphorylated of and at and 1 This panel samples of the within and are used for single-cell For barcodes using Scheme 3, we converted the DNA barcodes into antibody barcodes designed to three proteins and at and the DNAs used were for the in order to and the can be found in the Supporting 1. The is similar to a proteins from antibodies were by antibodies and For both cases, from DNA across the chip CVs that were with those of the underlying DNA barcodes (from for 2 and for Scheme 3, Figure 3 shows profiles of the signal intensities with the and demonstrate a uniformity for barcodes according to Scheme 2. we found that Scheme 3 produce barcodes that were in quality to those of Scheme 2, the consistency of Scheme 3 is to to its use of the and Moreover, Scheme 3 is the detailed procedure is more Scheme 2 can be Thus, we Scheme 2 as the barcode patterning Scheme 2 over of the patterned good quality for the test. from performed on a substrate according to a) Scheme 2 and b) Scheme 3 a protein and is with up to ten proteins (for Scheme 2 ). The to the of the barcode so that each two measurements of each is in because barcode pattern was employed. proteins were across five discrete channels per concentration for and four discrete channels per concentration for quantitative for analysis was from all the in each of the channels. By DEAL followed by standard protein the was The signal patterns within individual channels and between channels of similar demonstrate the good uniformity and quality of DNA barcodes. intensity profiles from analysis channel per concentration are in 2 We the use of the antibody barcodes by towards the multiplex of cytoplasmic proteins from single cells. is a significant of that demonstrates that cells can significant that be by proteomics techniques that across a We designed a highly microfluidic device of of cells in with a of antibody barcodes designed to proteins (Figure Supporting Information). Figure a shows a of the device and the protein The small the number of protein molecules thereby such a panel of proteins not be a high density antibody such as the barcodes utilized for the all the barcodes into such a small for in such a is it is to have consistent DNA loading across the are to be a) of the protein analysis or cells are in an proteins are assayed by introducing a the proteins to the DEAL barcode within the for for and DNA barcode array converted into DEAL antibody c) images of barcode assays the of using Scheme 2 Scheme 1 in to those which were using Scheme 2 barcodes. d) fluorescence intensity from the single-cell of We the as a model for our is the most brain found in and is the most of all As the exhibits biological and clinical is an and Thus, we assayed for proteins associated with the We representative of for protein from the of to five cells b and proteins were from single-cell and up to proteins were from five cells when using barcodes patterned by Scheme 2 b, whereas only protein be from barcodes by Scheme 1 (Figure the protein assays were for (Figure for the where proteins were for each protein were (Figure detailed analysis of as well as is We a protocol for generating DNA barcode patterns by comparing three microfluidics-based patterning schemes. We through both and that the electrostatic between PLL and the DNA backbone induces significant in the patterning but that those electrostatic interactions may be by DMSO to the resulting in uniform and highly reproducible barcodes patterned using long channels that barcodes across an entire glass covalent immobilization yields good ultimate but is by a relatively chemistry that limits DNA barcodes were with the DEAL technique to generate antibody barcodes, and then into designed microfluidic chips for assaying proteins from single and model cancer cells. of a panel of such proteins the of our platform to be applied to various biological clinical applications. for DNA PDMS chips were by The was using a negative with or an by a reactive The has long meandering channels with a The from channel to channel is which 10× higher density than standard, spotted PDMS and were in a onto the and 1 The PDMS was from the were and the device was onto a PLL or aminated glass to form channels. The number of microfluidic channels the of the microchannels were used in this of DNA For the DNA filling a DNA labeled with fluorescence on the in a 1:1 mixture of 1×PBS and DMSO or a 1:1 mixture of 1×PBS and water was The final DNA concentration was DNA solution was into the channel a constant pressure after the channels were fluorescence images were obtained by microarrays were using aminated substrates. Poly(amidoamine) (PAMAM) dendrimers in were 1:1 with in After of the activated dendrimers were introduced to the microfluidic channels, and to flow a to dendrimers, the channels were filled with in with After aminated DNA in 1×PBS were introduced to the channels and to flow the microfluidic device was from the substrate, and the was with that were not used immediately were in a generate the DNA barcode array for and single-cell DNA solutions are in the Supporting in 1×PBS were with DMSO and flowed into each of the microfluidic channels (Scheme 2 ). For Scheme 1 DNA solutions in 1×PBS were The chip was in a the solvent evaporated only DNA molecules the PDMS was from the glass substrate and the DNAs were to the PLL by thermal treatment The was with water to use in order to remaining from the solution evaporation step. for The PDMS microfluidic chip for the was by A was utilized with a layer with a flow The for the layer and the flow layer were with negative and positive respectively. The patterns for the flow layer were via thermal treatment. The layer was with a mixture of PDMS A and and the flow layer was by a mixture of A and on the flow layer were 1 the layer was from its and to the flow thermal treatment 1 that the two into a which was then from its and to the PDMS chip was to the DNA glass to form the The was in with cells were for 1 and then by they were introduced into the assays were by the chip with 3 in PBS to non-specific This 3 solution was used as a for most subsequent After a containing all (Scheme 2 or three (Scheme 3 in was flowed through the channels for 1 The were with proteins were flowed through the microfluidic channels for 1 These were followed by a containing antibodies in and a mixture of 1 and ssDNA in to the DNA is used for a The microchannels were with more the PDMS chip was the microarray was with and was to and from The procedure described was slightly for protein the chip was with a 3 followed by a containing all Supporting 2) in flowed for 1 were with The was loaded into the channels while 1 in Figure a) was by constant cells were introduced to the loading channels and microfluidic in Figure a) were by constant this the channels into After were were to allow of to the containing different of cells. The was performed on for two After that, the were and the was by the a containing antibodies in was flowed into the chip for 1 on followed by a mixture of and ssDNA in to the the microchannels were with the PDMS chip was and the microarray was with The of the chip and used for different solutions were described in Figure The microarray was with the to a fluorescence image of both and channels. were performed with the same of and and The fluorescence intensities for all barcodes in each were obtained and to the number by or Molecular The MD simulations were performed with the using the As an a single of DNA base were was using the DNA sodium ions were to the negatively charged phosphate groups on the DNA backbone. this is in a solvation of water DMSO molecules or 2) only water We used model to the water We performed 3 ns NPT MD simulations using with a time of and with a of The last 1 ns trajectory is employed for the analysis. the electrostatic the was employed using an of This was by and by a from the P and support from the through of by the of to are as are but not or are as by the The is not for the or of by the than be to the corresponding for the