This work was supported by the National Natural Science Foundation of China (32071399 and 62175071), the Science and Technology Program of Guangzhou (2019050001), the Guangdong Basic and Applied Basic Research Foundation (2021A1515011988), and the Open Foundation of the Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, China (JYG2009).

1. Fabrication of Au@CDs
NOTE: Figure 1 illustrates a fabrication procedure for Au@CDs.
<ol>
	<li>Prepare CD solution using citric acid (CA) and gallic acid (GA) via a typical hydrothermal treatment procedure18. Add 100 &#181;L of 3.0 mg mL-1 of the prepared CD solution into 200 &#181;L of 10 mM chloroauric acid (HAuCl4) (see Table of Materials) at room temperature for 10 s until a purple suspension is produced.</li>
	<li>Centrifuge the purple suspension at 4,000 &#215; g for 10 min at room temperature and gently remove the supernatant using a pipette if removing the Au@CD colloids is difficult.</li>
	<li>Resuspend the Au@CDs with 200 &#181;L of deionized water (resistivity of 18.2 M&#8486;cm) to wash the excess CDs.</li>
	<li>Repeat step 1.2 and obtain the core-shell NPs, redispersed in 100 &#181;L of deionized water, and store at 4 &#176;C. The NPs can be stored for 1 month.</li>
</ol>
2. Characterization of Au@CDs
<ol>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Leave the CD and Au@CD samples overnight at -80 &#176;C to lyophilize from a frozen solution and crush after lyophilization into powder.</li>
		<li>Disperse the obtained powder samples into the deionized water adequately by sonication, at 100% power, for 5 min.</li>
		<li>Drop a few drops of the sample suspension onto a Cu-coated TEM grid covered with a lacey carbon film, and capture images after drying using a transmission electron microscope at a 200 kV acceleration voltage (see Table of Materials).</li>
	</ol>
	</li>
	<li>Fourier transform infrared spectroscopy (FT-IR)
	<ol>
		<li>Repeat step 2.1.1 to obtain power samples, grind a few pre-dried KBr particles into powders in a mortar, add a pinch of the sample, and mix with the KBr powders simultaneously for IR characterization16.</li>
	</ol>
	</li>
	<li>Ultraviolet-visible-near infrared (UV-vis-NIR) absorbance spectroscopy
	<ol>
		<li>Prepare the suspension of CDs and Au@CDs with proper concentration to ensure the absorbance is less than one and perform UV-vis-NIR characterization16.</li>
	</ol>
	</li>
	<li>Raman spectra
	<ol>
		<li>Switch on the 785 nm semiconductor laser, with a laser power of 10 mW and objection magnification of 20x. Set the exposure duration to 5 s and accumulations number to three.</li>
		<li>Add an equal volume of prepared Au@CDs samples into 5 &#181;L of methylene blue (MB) solution and thoroughly mix.</li>
		<li>Place a drop of suspension onto the brass substrate to collect the SERS spectra.</li>
	</ol>
	</li>
</ol>
3. Cell culture
<ol>
	<li>Culture human epithelial lung carcinoma cells (A549 cells, passaged in the laboratory) in Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and incubate in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>Seed the cells in 96-well plates (1 &#215; 105 cells/well) and treat with Au@CDs in different concentrations in the range of 20-100 &#181;M. Use a CCK-8 assay kit to measure the cell viability (see Table of Materials). Three independent duplicates are used in each treatment.</li>
	<li>For performing the SERS experiments, proceed following the steps below:
	<ol>
		<li>Put a sterile sapphire chip (see Table of Materials) into the 12-well plates and then seed the cells onto a single well with 1 mL of medium. Incubate the plates in a humidified incubator at 37 &#176;C with 5% CO2overnight to reach 70%-80% confluence.</li>
		<li>Add the Au@CDs into the single well and incubate for 4-6 h. 
		NOTE: Before incubation, test the stability of substrates, especially solution phase NPs. These materials are susceptible to degradation through dissolution, aggregation, and sedimentation processes during storage and use, which may decrease EM losses and decrease SERS activity. More significantly, for cellular uptake, it could be largely affected by the size of the NPs22.</li>
		<li>During the incubation, substrates will enter the cells through endocytic uptake. After incubation, observe the cells under an optical microscope until a few black particles are seen inside the cells. 
		NOTE: If the SERS intensity is weak, try to enhance the Au@CD concentration or prolong the incubation time.</li>
		<li>Remove the medium, gently rinse the sapphire chips with phosphate-buffered saline (PBS), and dip them in PBS for SERS detection16.</li>
	</ol>
	</li>
</ol>
4. Cell SERS experiments
<ol>
	<li>Open the computer, switch on the Raman spectrometer, start the software and then switch on the 785 nm laser&#160;(see Table of Materials).</li>
	<li>Click on New Measurement and start a new spectral acquisition. Calibrate17&#160;with silicon wafers before sample measurement.</li>
	<li>Take a 20x objective lens to ensure a clear cellular image is observed, then change the objective lens to 50x. Set the laser power from low to high and the appropriate exposure time and accumulations. 
	NOTE: Before SERS measurement, check the appropriate laser power to prevent irreversible cell damage and ensure that the acquisition parameters are consistent. SERS intensity is related to laser power, spot size, accumulation, and exposure time.</li>
	<li>Choose a point of cells to measure and click on Run. Each cell is measured by 20 spots, and 10 cells are measured to take the average. 
	NOTE: Intracellular components are complicated, leading to wide spectra disparity. Therefore, take spectra in similar cellular regions and take as many spectra as possible.</li>
	<li>Save the spectra.</li>
	<li>Click on New Streamline image acquisition, then click on Video Review, select the range to be photographed, and click OK. Set the parameters: 785 nm edge streamline, centre of 1,200 cm-1, exposure duration of 5 s, accumulations number of three, and laser power to 50%.</li>
	<li>Click on New of Living imaging, choose Signal to Baseline, set the range from first limit 625 to second limit 1,700, and then click on Area setup. Then set the proper steps, and click on Apply and OK. Finally, click Run to start performing SERS imaging.</li>
</ol>
5. Bacterial culture and SERS measurements
<ol>
	<li>Dilute overnight cultures of E. coli and S. aureus (1:100) in 5 mL of the new Luria-Bertani (LB) medium (see Table of Materials). After growth at 37 &#176;C to A600 0.4 to 0.6,&#160;centrifuge the culture at 5,000 &#215; g for 5 min to pellet the cells.</li>
	<li>Resuspend the pellets in 1 mL of PBS followed by a 5 min 5,000 &#215; g centrifugation (at room temperature) twice.</li>
	<li>Mix the bacterial culture with the Au@CDs and observe the mixture directly under the SERS platform.</li>
</ol>
6. Data analysis
<ol>
	<li>Smooth the spectra and correct the baseline.</li>
	<li>Perform principal component analysis (PCA)16&#160;with the processed data.</li>
</ol>

Label-Free SERS Analysis: Au@Carbon Dot Nanoprobes for Molecular Fingerprinting

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The authors have nothing to disclose.

In summary, Au@CDs with an ultrathin CD shell of 2.1 nm have been successfully fabricated. The nanocomposites show superior SERS sensitivity than pure Au NPs. Also, Au@CDs possess excellent performance in reproducibility and long-term stability. Further research includes taking Au@CDs as substrates to perform SERS imaging of A549 cells31&#160;and to detect two bacterial strains32. It has been proved that Au@CDs can be used as an ultrasensitive SERS probe mainly based on the...

Single-cell analysis is essential for the study of revealing cellular heterogeneity and assessing the comprehensive state of the cell. The cell&#39;s instant response to the microenvironment also warrants single-cell analysis1. However, there are some limitations to the current techniques. Fluorescence detection can be applied to single-cell analysis, but it&#39;s limited by low sensitivity. Other challenges arise from the complicated fluorescence background of cells and the fluorescence photobleaching under long-term irradiation2. Surface-enhanced Raman scattering (SERS) may qualify in terms of single-cell analysis owing to its advantages, including (1) reflecting the intrinsic molecular fingerprint information and the instantaneous situation, (2) ultrahigh surface sensitivity, (3) convenient multiplex detection, (4) high photostability, (5) detection can be quantified for comparative analysis, (6) avoiding cellular autofluorescence with the NIR wavelength excitation, (7) detection can be performed in a cellular aqueous environment, and (8) detection can be directed to a specific region within the cell3,4,5.
There are two broadly recognized mechanisms to understand SERS as a fundamental phenomenon: electromagnetic enhancement (EM) as a dominant reason and chemical enhancement (CM). EM refers to, in a given frequency of the exciting field, the oscillation of collective electrons driven by electromagnetic waves when the frequency of the incident light matches the frequency of free electrons oscillating in the metal, giving rise to surface plasmon resonance (SPR). When localized SPR (LSPR) occurs through the incident laser impinging at the metal nanoparticles (NPs), it leads to the resonant absorption or scattering of the incident light.&#160;Consequently, the surface electromagnetic field intensity of metal NPs can be enhanced by two to five orders4. However, the key to the huge enhancement in SERS is not a single metal NP, but the gap between two NPs, which creates hot spots. CM is generated from two sides, including (1) interactions between target molecules and metal NPs and (2) target molecules being able to transfer electrons to/from metal NPs4,5. More exhaustive details can be found in these review articles4,5. Several promising methods for SERS biosensing and imaging in living cells have been presented in previous literature, for example, the detection of apoptotic cells6, proteins in organelles7, intracellular miRNAs8, cellular lipid membranes,9cytokines10, and metabolites11&#160;in living cells, as well as the identification and monitoring of cells by confocal SERS imaging2,11,12,13. Interestingly, label-free SERS presents the unique advantage of SERS, which can describe internal molecular spectra5.
A major issue for label-free SERS is a rational and reliable substrate. Typical SERS substrates are noble metal NPs because of their excellent capacity to scatter a lot of light14. Nowadays, more and more attention is paid to nanocomposites due to their remarkable physical and chemical properties and biocompatibility. More significantly, nanocomposites can show better SERS activity because of the intense EM induced by the hot spots on the nanohybrids and additional chemical enhancement originating from other non-metal materials15. For example, Fei et al. used MoS2quantum dots (QDs) as reducers to synthesize Au NP@MoS2 QD nanocomposites for label-free near-infrared (NIR) SERS imaging of mouse 4T1 breast cancer cell (4T1 cells)16. Also, Li et al. fabricated a 2D SERS substrate consisting of Au NPs and 2D hafnium ditelluride nanosheets for label-free SERS measurements of foodborne pathogenic bacteria17. Recently, carbon dots (CDs), good electron donors, have been used as reductants without other reductants or irradiation to synthesize Au@carbon dot nanoprobes (Au@CDs)18, which have been reported to be efficient materials to enhance SERS activity based on the charge-transfer (CT) effect between Au cores and CD shells19,20. More than that, CDs are recognized as the capping agent and a stabilizer to prevent Au NPs from aggregating21. In addition, it opens up more possibilities for reactions with analytes, as it can provide a large number of binding and active sites20. Taking advantage of the above, Jin et al. developed a fast and controllable method for fabricating Ag@CD NPs with unique SERS properties and excellent catalytic activities for monitoring heterogeneous catalytic reactions in real time18.
Herein, a facile and low-cost method for fabricating core-shell Au@CD SERS substrates to identify cellular components and label-free SERS live cell bioimaging, as well as to detect and differentiate Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was demonstrated, which holds promise for the early diagnosis of disease and a better understanding of cellular processes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x PBS buffer (Cell culture)</td>
			<td>Langeco Technology</td>
			<td>BL316A</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well cell culture plate</td>
			<td>LABSELECT</td>
			<td>11110</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8 (CCK-8)</td>
			<td>GLPBIO</td>
			<td>GK10001</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Shanghai Aladdin Biochemical Technology</td>
			<td>C108869</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Technologies</td>
			<td>3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant temperature magnetic agitator</td>
			<td>Sartorius Scientific Instruments</td>
			<td>SQP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic high speed centrifuge</td>
			<td>Shanghai Boxun</td>
			<td>SW-CJ-2FD</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose cell culture medium</td>
			<td>Procell</td>
			<td>PM150210</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Sartorius Scientific Instruments</td>
			<td>SQP</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme marker</td>
			<td>Thermo Fisher Technologies</td>
			<td>3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Zhejiang Tianhang Biological Technology</td>
			<td>11011-8611</td>
			<td></td>
		</tr>
		<tr>
			<td>Figure 1</td>
			<td>Figdraw.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fourier infrared spectrometer</td>
			<td>Thermo, America</td>
			<td>Nicolet 380</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>Tecan</td>
			<td>Infinite F50</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>Shanghai Aladdin Biochemical Technology</td>
			<td>G104228</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld Raman spectrometer</td>
			<td>OCEANHOOD, Shanghai, China</td>
			<td>Uspectral-PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td>HAuCl4</td>
			<td>Guangzhou Pharmaceutical Company (Guangzhou)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High resolution transmission electron microscope</td>
			<td>Thermo Fisher Technologies</td>
			<td>FEI Tecnai G2 Spirit T12</td>
			<td></td>
		</tr>
		<tr>
			<td>High temperature autoclave</td>
			<td>Shanghai Boxun</td>
			<td>YXQ-LS-50S&#160;<img alt="Equation 2" src="/files/ftp_upload/65524/65524eq02.jpg" style="margin: auto;" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nanjing Jiangnan Yongxin Optical</td>
			<td>XD-202</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth BR</td>
			<td>Huankai picoorganism</td>
			<td>028320</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical ultra-low temperature refrigerator</td>
			<td>Thermo Fisher Technologies</td>
			<td>ULTS1368</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pancreatin Cell Digestive Solution</td>
			<td>beyotime</td>
			<td>C0207</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin streptomycin double resistance</td>
			<td>Shanghai Boxun</td>
			<td>YXQ-LS-50S&#160;<img alt="Equation 2" src="/files/ftp_upload/65524/65524eq02.jpg" style="margin: auto;" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Pure water meter</td>
			<td>Millipore, USA</td>
			<td>Milli-Q System</td>
			<td></td>
		</tr>
		<tr>
			<td>Raman spectrometer</td>
			<td>Renishaw</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire chip</td>
			<td>beyotime</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic water bath</td>
			<td>Changzhou Noki</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clean table</td>
			<td>Shanghai Boxun</td>
			<td>SW-CJ-2FD</td>
			<td></td>
		</tr>
		<tr>
			<td>Uv-visible light absorption spectrometer</td>
			<td>MADAPA, China</td>
			<td>UV-6100S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire 3.4</td>
			<td>Renishaw</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

label-free surface-enhanced raman scattering bioanalysis based on au@carbon dot nanoprobes

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular fingerprint information of biological samples, as well as its potential in single-cell analysis. This work aims to establish a simple strategy for label-free SERS bioanalysis based on Au@carbon dot nanoprobes (Au@CDs). Here, polyphenol-derived CDs are utilized as the reductant to rapidly synthesize core-shell Au@CD nanostructures, which allows powerful SERS performance even when the concentration of methylene blue (MB) is as low as 10-9 M, due to the cooperative Raman enhancement mechanism. For bioanalysis, Au@CDs can serve as a unique SERS nanosensor to identify the cellular components of biosamples (e.g., cancer cells and bacteria). The molecular fingerprints from different species can be further distinguished after combination with the principal component analysis. In addition, Au@CDs also enable label-free SERS imaging to analyze intracellular composition profiles. This strategy offers a feasible, label-free SERS bioanalysis, opening up a new prospect for nanodiagnosis.

Fabrication of the Au@CDs is illustrated in Figure 1. The CDs were prepared from CA and GA via a typical hydrothermal process18. Au@CDs were rapidly synthesized by reducing HAuCl4 by CDs in aqueous media at room temperature. The size and morphology of CDs and Au@CDs can be observed by TEM and high-resolution (HR)TEM23. The prepared CDs are monodispersed with small sizes of nearly 2-6 nm (<strong class="xfig"...

Watch this Scientific Journal Video about Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging at JoVE.com

Author Spotlight: Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging 

In this study, we developed a low-cost surface-enhanced Raman scattering (SERS)-based fingerprint nanoprobe with favorable biocompatibility to show label-free live cell bioimaging and detect two bacterial strains, showing in detail how to get SERS spectra of living cells in a non-destructive method.

Label-Free Surface-Enhanced Raman Scattering Bioanalysis Based on Au@Carbon Dot Nanoprobes

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular ...

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Bioengineering

1.4K Views.  South China Normal University. In this study, we developed a low-cost surface-enhanced Raman scattering (SERS)-based fingerprint nanoprobe with favorable biocompatibility to show label-free live cell bioimaging and detect two bacterial strains, showing in detail how to get SERS spectra of living cells in a non-destructive method.

Video: Author Spotlight: Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging 

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...