Analisis Protein pada Single Cell dengan Microfluidics

A. Outline
1. Pendahuluan
2. Microfluidic Elektroforesis
3. Microfluidic Cytometry
4. Microfluidic Probe
5. Microfluidic Based Mass Spectrometry

B. Pembahasan

1. Pendahuluan
Single cell analysis has aroused great interest with its remarkable ability to investigate cell-
to-cell het-erogeneity in large populations. However, probing protein information at single
cells resolution including quantity, interactions, and dynamics have been great challenges
as a result of the small size of cells, the complexity and a large concentration range of the
protein and the lack of genome-wide amplification method. Fortunately, microfluidics
capable of high throughput, high reproducibility, large parallelization, easy operability and
low-cost, has been emerging as a powerful platform for the analysis of single cell proteins.
In this review, we focus the recent advances of microfluidics in single cell resolution protein
analysis, particularly covering the following aspects: (1) microfluidic electrophoresis
(capillary electro-phoresis and gel electrophoresis); (2) microfluidic cytometry
(microfluidic flow cytometry, droplet based cytometry and image cytometry); (3)
microfluidic probe and (4) microfluidics based mass spectrometry.

Gambar 1. Hasil Statistika Publikasi Relevan dari Tahun 2000 sampai 2019 mengenai Single Cell dan
Microfluidic; dan Single cell, Microfluidic, dan Protein (sumber: https://sciencedirect.com)
2. Microfluidic Elektroforesis
Analisis protein pada single cell dengan teknik microfluid elektroforesis dibagi menjadi dua,
yaitu microfluidic capillary elektroforesis dan microfluidic gel elektroforesis. Capillary
electrophoresis (CE) is a separation technique with the advantages of high-efficiency
separations, easy coupling with other ultrasensitive detection methods and compatible inner
diameter with single cells, resulting in its big potential in separating, detecting and analyzing
a large number of contents of single cells. Microfluidic CE as the separation strategy has
many advantages, such as high throughput, reduced separation times, low sample
consumption and rapid dissipation of the Joule heating. Microfluidic based analytical
systems using electrophoresis also allow online separation and detection of multiple
components at the single cell level via automatically single cell injection and manipulation.
Microfluidic CE based single cell analysis involved on line lysis of individual cell, and then
simultaneous electrophoretic separation and identification of its intracellular contents.

Although the throughput is improved in microfluidic CE, it is still not scalable to a large
number of parallel electrophoresis as-says, which required to probe heterogeneity within a
population of single cells. Critically, the majority of the single cell protein analysis
technologies rely on antibodies to recognize and capture the specific protein. Such
antibodies based methods are enabling for pro-tein detection, however, suffering from
analysis accuracy, as antibody cross-reactivity with other similar proteins can generatefalse
positive signals. To address these current limitations, singlecell western blots employed a
microscope slide covered with athin photoactive polyacrylamide (PA) gel micro-patterned
(30mm) with high-density open-microwell arrays (6720 microwells).

Gambar 2. Chip Analisis Single Cell (sumber: https://sciencedirect.com)

3. Microfluidic Cytometry
Analisis protein pada single cell dengan teknik microfluidic cytometry dibagi menjadi tiga,
yaitu microfluidic flow cytometry, microfluidic droplet based cytometry, dan microfluidic
image cytometry. Flow cytometry (FCM)first appeared in the 1960s and is another most
widely used tool to characterize the surface targets of single cells (protein analysis) using
LIF technology, due to its over-whelming superiority of multiple protein feasibility and high
 throughput, but while flow cytometry is the common tool in immunology for detection,
sorting and collection of cells with high-throughput, which focuses on membrane protein
and intracellular protein analysis by directly probing the protein in single cells in a buffer
suspension, it is less fitting the measurement of the secreted molecule from single cells.

Microfluidic droplet based cytometry was modified to encapsulate single cells and reagents
in independent aqueous pico- or nanoliter droplets dispersed in an immiscible carrier oil,
allowing the quantification of target proteins secreted bysingle cells. A particular advantage
of microfluidic droplet based cytometry is that droplets provide a simple means of linking
genotype with phenotype through compartmentalization: encapsulating single cells that
produce an enzyme of interest in picolitre-volume water droplets together with afluorogenic
substrate.

Although above any mentioned microfluidic cytometer is aquick and high throughput
analytic method, it's still limited inspatial resolution and time-dependent analysis because
of theirreliance on single end point interrogation strategy. To address suchissues,
microfluidic image cytometry (MIC) offers an opportunity to design high-efficiency
platforms that combines the traditionalhigh-throughputflow cytometry with the spatial
resolution optical microscopy.

4. Microfluidic Probe
Microfluidic probe (MFP) was an alternativepowerful approach for in situ single cell protein
analysis in the open space without walls, especially for adherent cells and tissue slices. It
combineshydrodynamically confinedflow with the concept of scanningprobes for surfaces
processing, which could be used to createchemical gradients on surfaces, protein
microarrays, localized staining of cells. Lately, this promising tool was applied to monitor
the protein activities from an amountof bulk-level cell lysate, even from a single cell
selected from anadherent cell population. However, they were stillsuffered from low
throughput, requiring manual handling of mi-cromanipulators and precise device
positioning and thefluidflowmay be susceptible to disturbances and variations.

5. Microfluidics Based Mass Spectometry

Mass spectrometry (MS) has been the major analytical platform for single-cell metabolite
analysis because of its high sensitivity, selectivity and multiplexing capabilities. In
comparison, there are still lots of challenges for single cell protein analysis by MS: i) the
large number and great diversity of proteins presented in most cells, large dynamic range of
protein abundance, existing lots of post-translational modification including
phosphorylation; ii) difficulty in sample processing steps including
extraction/solubilization, purification, separation, reduction of disulfide bonds, alkylation
of free cysteine residues and digestion, and the potential sample losses during the whole
course; iii) requiring intricate data analysis and post-processing.
 To further increase sample processing efficiency and improve proteomic sensitivity, some
emerging novel methods were introduced, including nanoflow chemical separations, high
performance ion optics and detectors, low-loss sample preparation and microfluidics.
Among them, microfluidic devices coupled to MS have emerged as a powerful platform for
single cells proteomics measurement with the ability to manipulate and dispense ultra-small
volumes (femto-to nanoliters), to enhance overall sensitivity by significantly minimizing
protein and peptide losses using auto-mating sample pretreatment steps with the potential
for high-throughput parallel analysis.

Daftar Pustaka

Chen, et al. 2019. Microfluidics towards Single Cell Resolution Protein Analysis di
https://sciencedirect.com/science/article/pii//S0165993619301657/ , diakses pada 16 Oktober
2019.

Wu, et al. 2004. “Chemical Cytometry on a Picoliter-Scale Integrated Microfluidic Chip”

dalam Proceedings of the National Academy of Sciences of the United States of America, Vol
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Krutzik, et al. 2006. Fluorescent Cell Barcoding in Flow Cytometry Allows High-Throughput
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pada 16 Oktober 2019.

Juncker, et al. 2005. Multipurpose Microfluidic Probe di

https://www.nature.com/articles/nmat1435?proof=trueJul , diakses pada 16 Oktober 2019.
Lee, et al. 2009. Microfluidic Chips for Mass Spectometry-Based Proteomics di
https://onlinelibrary.wiley.com/doi/abs/10.1002/jms.1585 , diakses pada 16 Oktober 2019.