Multiplex Profiling of Biomarker and Drug Uptake in Single Cells Using Microfluidic Flow Cytometry and Mass Spectrometry

Abstract

To perform multiplex profiling of single cells and eliminate the risk of potential sample loss caused by centrifugation, we developed a microfluidic flow cytometry and mass spectrometry system (μCytoMS) to evaluate the drug uptake and induced protein expression at the single cell level. It involves a microfluidic chip for the alignment and purification of single cells followed by detection with laser-induced fluorescence (LIF) and inductively coupled plasma mass spectrometry (ICP-MS). Biofunctionalized nanoprobes (BioNPs), conjugating ∼3000 6-FAM-Sgc8 aptamers on a single gold nanoparticle (AuNP) (K_d = 0.23 nM), were engineered to selectively bind with protein tyrosine kinase 7 (PTK7) on target cells. PTK7 expression induced by oxaliplatin (OXA) uptake was assayed with LIF, while ICP-MS measurement of ¹⁹⁵Pt revealed OXA uptake of the drug in individual cells, which provided further in-depth information about the drug in relation to PTK7 expression. At an ultralow flow of ∼0.043 dyn/cm² (20 μL/min), the chip facilitates the extremely fast focusing of BioNPs labeled single cells without the need for centrifugal purification. It ensures multiplex profiling of single cells at a throughput speed of 500 cells/min as compared to 40 cells/min in previous studies. Using a machine learning algorithm to initially profile drug uptake and marker expression in tumor cell lines, μCytoMS was able to perform in situ profiling of the PTK7 response to the OXA at single-cell resolution for tests done on clinical samples from 10 breast cancer patients. It offers great potential for multiplex single-cell phenotypic analysis and clinical diagnosis.

Keywords: biofunctionalized nanoprobes; flow cytometry; mass spectrometry; microfluidic chip; multiplex profiling; single-cell heterogeneity.

Scheme 1. Working Principles of the Microfluidic System for Single Cell Alignment/Purification in the LIF/ICP-MS Assays

After OXA exposure, the trypsinized cell suspension was incubated with BioNPs to enable the capture of these cells by the probe. The cells conjugated with BioNPs were then aligned and purified in the microfluidic chip. Finally, the single cells were detected with LIF and introduced to TRA-ICP-MS to perform efficient background-free multiplex single-cell analysis.

Figure 1

Fabrication and characterization of BioNPs. (A) TEM images and (B) AFM images of the BioNPs nanoprobes along with their size distribution. (C) Nanoparticle tracking analysis (NTA) confirmed that AuNPs were conjugated with Sgc8 aptamers. (D) The evaluation of the dissociation constants of BioNPs and Sgc8-AuNPs to MCF-7 cells. (E) Scrutinizing the treatment time of BioNPs nanoprobes conjugating on MCF-7 cells. Incubation of the probe with the cells can be completed in 20 min. DAPI, DiI, and 6-FAM represent the nucleus, cell membrane and BioNPs probe, respectively. (F) The relative fluorescence intensity of BioNPs conjugated on various cell lines. (G) The relative expressions of PTK7 in various cell lines by real-time PCR (qPCR). The data indicate mean ± standard deviation (SD). Three independent groups tested by using biological replicates (n = 3) were used for each data point.

Figure 2

Evaluation of OS-Chip. (A) Fluid velocity field of nine cross sections (1–9) in the y-axis along the micropillar-HcC-micropillars at a flow rate of 10 μL/min and Re = 1.01. (B) F_co values in the D3-ESC configuration under various flow rates of 5, 10, 20, 50, 100, 300, 500, 800, 1000, and 1200 μL/min (Re = 0.51, 1.01, 2.01, 5.04, 10.07, 30.21, 50.35, 80.57, 100.70, and 120.80). (C) Simulation results of the single-cell trajectory achieved in the purifier with P2 configuration. (D) The actual flow rates in Outlets 1 and 2. An independent t-test is performed to obtain statistical pairwise differences between Outlets 1 and 2 (***p < 0.001). (E) Evaluation of the purification performance for the P2 by using different concentrations of AuNPs suspension collected from the Outlet 2. Residual Rate represents residual quantity of AuNPs in the collected medium from Outlet 2. (F) The comparison of transport efficiency or measurement efficiency for the single cells between the vertical and horizontal sampling modes. (G) Photograph of the OS-Chip with two dyes (blue and red) loading in two inlets (Inlet 1 and Inlet 2). Shear stress gradient (H) and shear stress of the whole cell path (I) in the microfluidic chip. Three independent chips prepared by using biological replicates (n = 3) were used for each data point.

Figure 3

Analytical performance of μCytoMS. (A) Photograph of the μCytoMS system. The inset represents the frequency histogram and Gauss fit of the peak for single fluorescent particle signals. It indicates that the system is able to quantify fluorescent response of individual particles. ICP-MS temporal profile (B) and intensity distribution (C) of ¹⁹⁷Au spikes of AuNPs solution for the detection of single BioNPs probes (n = 5, the confidence limit is 95%). (D) Fluorescence burst data of single Apt-AuNPs probes during a 5 s time window achieved from LIF. (E) Frequency histogram of peak height of single BioNPs probe Gauss fit from LIF. (F) Heatmap of the number of probe labeling on 90 single-cells from nine cell lines. (G) ROC curves showing the high accuracy (AUC = 1) of the SUM signature in cell phenotyping. SUM means integrated dual-mode detection of LIF and ICP-MS with higher accuracy than LIF (AUC = 0.891) or ICP-MS (AUC = 0.383) alone for cell analysis. (H) Close-up of the green and blue triangles marked signals in Figure S30A,B, along with LIF (red line) and ICP-MS (gray line) data coincidence after correction with the Correction time. (I) Content of the OXA and the fluorescence burst data of the same 5 single-cells from nine cell lines. (J) LDA canonical score plots for the response of the BioNPs assemblies to single cells from nine cell lines. Three independent groups to be tested by using biological replicates (n = 3) were used for each data point.

Figure 4

Evaluation of performance of μCytoMS on clinical samples. (A) Quantification of CTCs in 50 μL of blood samples from patients and healthy controls. (B) The numbers of CTCs were detected respectively from 50 μL of cancer patient blood with detection by LIF and ICP-MS. (C) The scatter plot for μCytoMS profiling of individual patient CTCs. (D) LDA canonical score plots for the response of the BioNPs assemblies to single CTCs from ten patients. The data indicate mean ± standard deviation (SD). Three independent groups to be tested by using biological replicates (n = 5) were used for each data point. The Student’s t test was used to compare the means of two groups (A).