Article Cite This: ACS Sens. XXXX, XXX, XXX−XXX
pubs.acs.org/acssensors
Hollow Nanoneedle-Electroporation System To Extract Intracellular Protein Repetitively and Nondestructively Gen He,†,§ Chengduan Yang,†,§ Tian Hang,† Di Liu,‡ Hui-Jiuan Chen,† Ai-hua Zhang,† Dian Lin,† Jiangming Wu,† Bo-ru Yang,† and Xi Xie*,† †
ACS Sens. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/08/18. For personal use only.
The First Affiliated Hospital of Sun Yat-Sen University; State Key Laboratory of Optoelectronic Materials and Technologies, School of Electronics and Information Technology, Sun Yat-Sen University, Guangzhou 510006, China ‡ Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637, United States S Supporting Information *
ABSTRACT: Techniques used to understand the dynamic expression of intracellular proteins are critical in both fundamental biological research and biomedical engineering. Various methods for analyzing proteins have been developed, but these methods require the extraction of intracellular proteins from the cells resulting in cell lysis and subsequent protein purifications from the lysate, which limits the potential of repetitive extraction from the same set of viable cells to track dynamic intracellular protein expression. Therefore, it is crucial to develop novel methods that enable nondestructive and repeated extraction of intracellular proteins. This work reports a hollow nanoneedle-electroporation system for the repeated extraction of intracellular proteins from living cells. Hollow nanoneedles with ∼450 nm diameter were fabricated by a material deposition and etching process, followed by integration with a microfluidic device. Long-lasting electrical pulses were coupled with the nanoneedles to permeate the cell membrane, allowing intracellular contents to diffuse into the microfluidic channels located below the cells via hollow nanoneedles. Using lactate dehydrogenase B (LDHB) as the model intracellular protein, the nanoneedle-electroporation system effectively and repeatedly extracted LDHB from the same set of cells at different time points, followed by quantitative analysis of LDHB via standard enzyme-linked immunosorbent assay. Our work demonstrated an efficient method to nondestructively probe intracellular protein levels and monitor the dynamic protein expression, with great potential to help understanding cell behaviors and functions. KEYWORDS: hollow nanoneedles, electroporation, intracellular protein, extraction, dynamic analysis n the field of biomedical research, protein detection and analysis have emerged as critical techniques in understanding cellular functions and physiological activities, and have become important tools in early disease diagnostics and drug development.1−3 Intracellular proteins play critical roles in tuning cellular activities and controlling cellular functions such as cell division, differentiation, and apoptosis.4−6 The expression of intracellular proteins varies according to the cell cycle and is altered by environmental stimulants.7−9 Understanding the dynamical expression of intracellular proteins has attracted tremendous research interest in fundamental biological research as well as in biomedical engineering. Multiple techniques including enzyme-linked immunosorbent assay (ELISA), Western blot, and mass spectrometry have been developed to analyze proteins. These methods have achieved great success but they rely on the pre-extraction of proteins from cells.10−12 Currently, the most widely used protein extraction technique is the direct lysis of cells, followed by protein purification from the cell lysates.13,14 However, protein extraction based on cell lysis eliminates the possibility of repeated extraction from living cells. Protein extraction from
I
© XXXX American Chemical Society
cell lysates only allow protein sensing at a single time point rather than dynamic monitoring across multiple time points. Due to the heterogeneity of individual cells even within the same cell type, cell lysis lack the capability to probe dynamic intracellular protein expression.15,16 Other than cell lysis, another intracellular protein sensing technique employs fluorescent nanomaterials or molecules to interact with target intracellular proteins, and display responsive optical signals for detection (such as through fluorescence resonance energy transfer).17,18 However, endosomal trapping of nanomaterials has greatly limited the accessibility of cytosolic proteins, and the accumulation of nanomaterials within living cells decreases cell viability.19−21 Furthermore, single nanoprobe systems prepared via advanced nanofabrication have been reported to penetrate the cell membrane and to detect intracellular electrical signals Received: May 5, 2018 Accepted: August 27, 2018 Published: August 27, 2018 A
DOI: 10.1021/acssensors.8b00367 ACS Sens. XXXX, XXX, XXX−XXX
Article
ACS Sensors
Figure 1. Schematic of the hollow nanoneedle-electroporation system, nanoneedle fabrication, and device integration. (a) Illustration of the hollow nanoneedle-electroporation system for nondestructive extraction of intracellular proteins from living cells. The electrical pulses locally permeated cell membrane above the nanoneedle, allowing intracellular proteins to diffuse out of the cells into the microfluidic channel underneath the cells, followed by analysis via standard ELISA. (b) Illustration of the fabrication procedure of hollow nanoneedles. (c) Illustration of the integration of hollow nanoneedle array with a microfluidic device. The fluidic channel serves as a reservoir to collect extracted cytosolic contents. A crosssectional view of the device is provided to clearly demonstrate the device structure.
diameter to enable higher protein diffusivity within the nanoneedle channels. We then coupled the nanoneedles with long-lasting electrical pulses to extend the protein extraction time. The nanoneedles were fabricated using multiple steps of material deposition and etching, and the final product was integrated with a microfluidic device. Cells remained viable when cultured on top of the nanoneedles and following the application of long-lasting electrical pulses. The electrical pulses locally porated the cell membrane above the nanoneedle, and this allowed intracellular proteins to diffuse out of the cells into the microfluidic channels located beneath the cells. Standard ELISA was used to analyze intracellular protein extraction efficiency (Figure 1a). Lactate dehydrogenase B (LDHB) was employed as the model intracellular protein. The hollow nanoneedle-electroporation system successfully extracted LDHB from the same set of cells at different time points. The extraction efficiency was significantly higher than the sole use of transient electric pulses. This work demonstrated an efficient strategy to nondestructively extract intracellular proteins to probe dynamic cell behaviors and functions with potential applications in early disease detection and drug development.
and biochemical signals, or to extract intracellular materials. 22−25 The size of the nanoprobes were generally significantly smaller than the cell size to avoid cell destruction during the extraction process.26,27 For example, Vorholt et al. developed fluidic force microscopy to quantitatively extract enzymes from a single cell.28 However, due to the complicated operational methodology used in single nanoprobe systems, these techniques were performed on individual cells, thus limiting high throughput analysis of a large number of cells. Whereas approaches based on arrays have the potential to massively parallelize the process to enable high throughput applications. Additionally, multiple nanowire arrays have been used to penetrate cells and to detect intracellular proteins in many cells simultaneously.29,30 Park et al. sandwiched cells between nanowire arrays to probe enzymatic activity.31 However, solid nanowires required preconjugation with antibody on its surface, which posed a challenge for repetitive applications on the same set of cells or multiplex protein detection. Here we developed a hollow nanoneedle-electroporation system for the nondestructive extraction of intracellular proteins from living cells. Hollow nanoneedles, also called nanostraws, have been previously used to deliver drugs and to extract intracellular proteins and mRNA from cells.32−34 Prior studies demonstrated that small nanostraws with diameter of 150 nm coupled with transient electric pulses were able to extract proteins. However, the aforementioned method demonstrated low extraction efficiency, and the analysis method coupled with standard ELISA to detect nonfluorescently labeled proteins required a large quantity of cells to meet the detection threshold of the ELISA kit.34 Alternatively, our work used large hollow nanoneedles with ∼450 nm
■
RESULTS AND DISCUSSION The nanoneedle array was fabricated using a track-etched polycarbonate membrane as the starting template.32,33 The initial pore size of the track-etched polycarbonate membrane was 150 nm with pore densities of 0.3 pores/μm2. The pores were enlarged to 450 nm via O2 plasma etching at 50 W for 4 min.35 Then, the membrane was deposited with 40 nm of aluminum oxide on all surfaces including the top and bottom surfaces as well as the inner pore sidewalls via atomic layer deposition B
DOI: 10.1021/acssensors.8b00367 ACS Sens. XXXX, XXX, XXX−XXX
Article
ACS Sensors
coated with poly-D-lysine solution to promote cell adhesion and culture. In this work, cell medium containing Chinese hamster ovary (CHO) cells was deposited into the top PDMS well. CHO cells that natively expressed green fluorescent protein (GFP) were used in this work to visualize and dynamically monitor cells via fluorescence microscopy. Following deposition, the CHO cells settled onto the nanoneedles. After 24 h cell culture, the nanoneedle-cell interface was examined using confocal fluorescence microscopy (Figure 2c). The nanoneedles were stained with fluorescent dye, Alexa Fluor 660 (red fluorescence). GFP cells on red fluorescent nanoneedles were fixed and imaged to visualize the interface between the nanoneedles and the cells. The cells were observed to spread on top of the nanoneedles, with cell bodies deformed and resting on the needle array. In addition, the cell-nanoneedle samples were fixed and prepared with critical point drying for SEM imaging (Figure 2d). The images showed that the cells deformed around the hollow nanoneedles and the cell membrane engulfed the nanoneedles. These results were consistent with other microscopic studies of nanowire-cell interface which found that cell membrane tend to fully deform and formed tight-junctions with 1-D nanowire objects given a sufficient cell culture period.26,27 An electric field could be localized on the cell membrane above each nanoneedle due to the close contact between the cell membrane and the nanoneedles. The close interface and uniform contact geometry allowed a large population of cells to be porated at a low voltage (
