assay sensitivity is an important factor here, which demands improvement on engineering design and surface chemistries of the microchip. A reliable measurement/decision protocol is also required to streamline the single-cell proteomic assays and to ensure a robust and highly reproducible data collection and analysis, so that meaningful comparison can be made between datasets collected across time points, patient samples and assay conditions. Additionally, a challenge with any clinical study that attempts to match patients with appropriate therapies and therapy combinations, and for which the disease is highly heterogeneous, is that we don’t know, prior to analysis, what drugs will be required, and so we need to design a trial that can potentially accommodate multiple drugs from different manufacturers and select the appropriate dose smartly.
In this chapter, we first discuss the advances in engineering and surface chemistry that address the technical challenges for translating SCBCs into the clinic. We further present some preliminary data collected from a pediatric GBM patient bearing an EGFR amplified tumor to demonstrate the workflow of the clinical translation.
5.2EXPERIMENTAL METHODS
5.2.1 Surface chemistry optimization for clinical applications
Primary cells are smaller in size and have a higher dynamic range in terms of oncogenic signaling compared to the genetic modified cell lines. An antibody microarray with high sensitivity is therefore critical in this context, which in turn requires a higher DNA loading for patterning the ssDNA microarray. To increase the loading and overall uniformity, we developed a method based upon covalent binding between ssDNA and poly-L-lysine (PLL) in place of the original evaporation method (See Chapter 2). An
additional PLL coating step is also included (Fig. 5.1).
Specifically, after bonding of PDMS device to the PLL slide, 0.1% PLL solution (Sigma Aldrich) is flowed through the microchannels followed by air blow drying. Then a library of amine modified ssDNAs, diluted in a mixture of DMSO and deionized water (v/v=3:2) with a final concentration of 300μM and mixed with 2mM BS3 solution (a linker molecule that contains an amine-reactive N-hydroxysulfosuccinimide (NHS) ester at each Figure 5.1 The reaction scheme for covalent DNA patterning. Accessible α-amine groups present on the amine-terminated ssDNA and ε-amines on lysine residues react with NHS-esters and form amide bonds. A covalent amide bond is formed when the NHS-ester crosslinking agent reacts with a primary amine releasing N -hydroxysuccinimide
end of an 8-carbon spacer arm, v/v=1:1), is flowed into each of the microfluidic channels.
The solution-filled chip is then placed in a sealed petri-dish with controlled moisture for 90 minutes to immobilize amine-terminated ssDNAs to the PLL surface. After incubation, the PDMS elastomer is removed from the glass slide in water containing 0.02% SDS followed with intensive washing in 0.02% SDS in water.
Fig. 5.2 shows how different surface chemistries will affect the DNA barcode patterning with respect to DNA loading and overall uniformity. Comparing with other methods, covalent binding method gives out highest loading and best over uniformity (lowest CV, Fig. 5.2 b). It also shorten the original 3-5 days process by evaporation method to only 1 day. The high DNA loading achieved plus other surface chemistry optimizations Figure 5.2. The surface chemistry of DNA barcode patterning, and its importance for quantitative single-cell protein immunoassays. a. The microfluidic flow patterning template used to prepare barcodes on poly-L-lysine(PLL)-coated glass slides. i. The elastomer flow patterning mold contains 1 channel for each barcode stripe – a mold for a 20 element barcode is drawn. The channels meander across the glass surface, and are on the order of 1 meter long and 10-20μm wide, depending upon the design. ii. ssDNA oligomers are initially patterned, and the quality of those DNA barcodes is assessed by hybridizing each strand with a complementary, dye-labeled ssDNA’ oligomer. iii. The digitized fluorescence micrograph shows the uniformity of a 10-element barcode, across the region indicated by the yellow bar in ii. b. Digitized fluorescence data reflects the DNA loading of 20μm wide barcode stripes, based on various patterning chemistries. “O” and “X” mean that the indicated chemistry was or was not used, respectively; CV (coefficient of variation) values through the entire slide were listed, showing the loading uniformity from various patterning strategies. c. Calibration data for the protein p- ERK, measured using the various chemistries. Note that surface chemistry improvements yield more than a 10-fold increase in assay sensitivity, enabling single cell assays of both highly challenging primary cells (the GBM patient sample), and model cell lines.
such as matching best antibody ELISA pairs and using brighter dyes finally transfer to a more than 10-fold increase in assay sensitivity and more than 50-fold in signal to noise ratio (Fig. 5.2 c).
5.2.2 High throughput solutions
Single-cell functional proteomic microchips are the diagnostic workhorse for clinical applications. The nature of the single-cell biology is that statistical numbers of single cells
must be analyzed for any given assay to generate a meaningful result2. Although our first microchip prototype contained only 120 microchambers3, the SCBC has been consistently developed and optimized since then and now it contains 320 microchambers per chip. Two chips have to be run in parallel each time to ensure enough statistics, which is not yet
Figure 5.3 Illustration of the two-layer cross-stripe DNA microarray construction. (a, top) Schematic illustration of the chemical patterning to produce the high density cross-stripe ssDNA arrays. Stripes of DNA are first patterned onto a PLL coated glass slide using a flow patterning mold . A second flow patterning step, orientated perpendicular to the first, is used to create unique addresses at the intersections of the cross stripes. The color coded DNA oligomers illustrate the patterning/hybridization sequence to produce the final array. (a, bottom) Validation of the cross-stripe barcode microarray. Each square unit of fluorescent spots represents many copies of a 3×3 array. The plot at right provides the fluorescence intensity profile of the vertical line through the two square unit. (b) The microfluidic flow patterning template used to prepare high density 10μm × 10μm cross-stripe DNA array throughout the entire slide by covalent method. It will generate more than 240,000 array spots in the whole slide after patterning.