Cells are the basic units of the structure and activities of living species, and single cell-based research is the foundation of life science. However, because of the tiny volume of cells, complex microenvironment within cells and the low concentrations of their components, single-cell analysis is still a challenging task in many aspects. Traditional cell studies have largely relied on ensemble measurements, and one of the challenges with these population-averaged analyses is that they only uncover the dominant biological traits in a population, whereas cell-to-cell differences (cellular heterogeneity) are greatly obscured. Single-cell analysis (at the DNA, RNA, protein or morphological level) offers the promise of unveiling cellular heterogeneity and its central relevance to biological functions.
Proteins are major effectors and regulators in cells. Besides, abnormal protein expression is frequently associated with diseases, such as cancer. Global protein expression analyses have revealed that many essential proteins are produced at low-copy-number levels (fewer than 1,000 molecules per cell) and these low-copy-number proteins perform multiple crucial functions in various biological processes. However, compared with the impressive advances in genome and transcriptome analysis in single cells, protein analysis in single cells lags severely behind because proteins cannot be amplified in the same way as DNA or RNA molecules. Therefore, analytical tools with superior sensitivity to enable the analysis of low-copy-number proteins in single cells are high in demand.
Recently, Professor Zhen Liu’s group at the State Key Laboratory of Analytical Chemistry for Life Science in our school reported an innovative technique called plasmonic immunosandwich assay (PISA), for detection of low-copy-number proteins in a single living cell (Angewandte Chemie International Edition, 2016, 55, 13215). Furthermore, this method has been extended for quantitative determination of subcellular microRNAs in single living cells (Chemical Science, 2018, 9, 7241) and disease biomarkers in clinical samples (Analytical Chemistry, 2016, 88, 12363；Analytical Chemistry, 2019, 2019, 91, 4831；Analytical Chemistry, 2019, 91, 9993；Biosensors and Bioelectronics, 2019, 145, 111729). Moreover, the PISA approach has also been applied to the measurement of dynamic signaling proteins and protein complexes in single cells after drug stimulation, demonstrating the application potential of the method in personalized medicine (Analytical Chemistry, 2020, 92, 12498).
The workflow of the single cell plasmonic immunosandwich assay (scPISA) is shown in Figure 1, and the principle is as follows: an affinity ligand-functionalized gold-based extraction microprobe is precisely inserted into a single living cell of interest through a three-dimensional manipulator. After a quick in vivo immunoaffinity extraction, the microprobe is taken out of the cell, washed to remove nonspecifically bound background molecules and then labeled with corresponding silver-based plasmonic nanotags. Thus, extraction microprobe/protein/plasmonic nanotag sandwich-like immunocomplexes are formed on the surface of the extraction microprobe, which are subsequently detected under a Raman microscope. High-performance antibodies and artificial antibodies (including molecularly imprinted polymers and nucleic acid aptamers) used in in vivo immunoaffinity extraction can ensure high specificity of the extraction. On the other hand, due to the plasmonic coupling effect (hot spot) between the silver-based nanotags and the surface of the gold-based extraction microprobe, upon radiation of a laser beam, the intensity of the surface-enhanced Raman scattering (SERS) signal of the plasmonic nanotag increases dramatically (the overall enhancement can be more than nine orders of magnitude), providing ultrasensitive detection at the single-molecule level. Instrumental platforms used for scPISA are shown in Figure 2.
Figure 1. The workflow of scPISA.
Figure 2. Instrumental platforms used for scPISA.
In contrast to current single-cell analysis techniques for protein detection, the scPISA approach has several advantages. First, scPISA has high specificity and single-molecule sensitivity (with a lower detection limit of about six protein molecules from a single living cell), which are two key factors enabling detection of low-copy-number analytes in the complicated cellular environment. Second, scPISA is fast, and the key steps from in vivo immunoaffinity extraction to Raman signal readout, require only ~6-15 min. Third, scPISA is a minimally invasive approach, which can retain a cell in its native state, and the cell can remain alive and be used for downstream experiments after analysis. Overall, the precision and flexibility of the scPISA approach provide a powerful tool with applications from basic research to clinical use.
The paper entitled “Probing low-copy-number proteins in single living cells using single-cell plasmonic immunosandwich assays” has been published in Nature Protocols on June 5, 2021 (https://doi.org/10.1038/s41596-021-00547-9). Dr. Jia Liu is the first author of this paper. Professor Zhen Liu is the corresponding author. This work was funded by Key Scientific Instrumentation Grant (21627810) from the National Natural Science Foundation of China, and Excellent Research Program of Nanjing University (ZYJH004).