8 research outputs found
Facile and Rapid Generation of Large-Scale Microcollagen Gel Array for Long-Term Single-Cell 3D Culture and Cell Proliferation Heterogeneity Analysis
Microfabricated devices are suitable
for single-cell analysis due
to their high throughput, compatible dimensions and controllable microenvironment.
However, existing devices for single-cell culture and analysis encounter
some limitations, such as nutrient depletion, random cell migration
and complicated fluid shear influence. Moreover, most of the single-cell
culture and analysis devices are based on 2D cell culture conditions,
even though 3D cell culture methods have been demonstrated to better
mimic the real cell microenvironment in vivo. To solve these problems,
herein we develop a microcollagen gel array (ÎĽCGA) based approach
for high-throughput long-term single-cell culture and single-cell
analysis under 3D culture conditions. Type-I collagen, a well-established
3D cell culture medium, was used as the scaffold for 3D cell growth.
A 2 × 2 cm PDMS chip with 10 000 μCGA units was
fabricated to encapsulate thousands of single cells in less than 15
min. Single cells were able to be confined and survive in ÎĽCGA
units for more than 1 month. The capability of large-scale and long-term
single-cell 3D culture under open culture conditions allows us to
study cellular proliferation heterogeneity and drug cytotoxicity at
the single-cell level. Compared with existing devices for single-cell
analysis, ÎĽCGA solves the problems of nutrient depletion and
random cellular migration, avoids the influence of complicated fluid
shear, and mimics the real 3D growth environment in vivo, thereby
providing a feasible 3D long-term single-cell culture method for single-cell
analysis and drug screening
supplemental results for R6G spectra and OTA titration from Facile fabrication of microfluidic surface-enhanced Raman scattering devices via lift-up lithography
R6G spectra on AuNP assembled area, and relative intensity of SERS signal as a function of the concentration of OTA
supplemental results for R6G spectra and OTA titration from Facile fabrication of microfluidic surface-enhanced Raman scattering devices via lift-up lithography
R6G spectra on AuNP assembled area, and relative intensity of SERS signal as a function of the concentration of OTA
Target-Responsive DNA Hydrogel Mediated “Stop-Flow” Microfluidic Paper-Based Analytic Device for Rapid, Portable and Visual Detection of Multiple Targets
A versatile point-of-care assay platform
was developed for simultaneous
detection of multiple targets based on a microfluidic paper-based
analytic device (ÎĽPAD) using a target-responsive hydrogel to
mediate fluidic flow and signal readout. An aptamer-cross-linked hydrogel
was used as a target-responsive flow regulator in the ÎĽPAD.
In the absence of a target, the hydrogel is formed in the flow channel,
stopping the flow in the ÎĽPAD and preventing the colored indicator
from traveling to the final observation spot, thus yielding a “signal
off” readout. In contrast, in the presence of a target, no
hydrogel is formed because of the preferential interaction of target
and aptamer. This allows free fluidic flow in the ÎĽPAD, carrying
the indicator to the observation spot and producing a “signal
on” readout. The device is inexpensive to fabricate, easy to
use, and disposable after detection. Testing results can be obtained
within 6 min by the naked eye via a simple loading operation without
the need for any auxiliary equipment. Multiple targets, including
cocaine, adenosine, and Pb<sup>2+</sup>, can be detected simultaneously,
even in complex biological matrices such as urine. The reported method
offers simple, low cost, rapid, user-friendly, point-of-care testing,
which will be useful in many applications
Simple and Rapid Functionalization of Gold Nanorods with Oligonucleotides Using an mPEG-SH/Tween 20-Assisted Approach
DNA conjugated gold nanorods (AuNRs)
are widely applied for nanostructure
assembly, gene therapy, biosensing, and drug delivery. However, it
is still a great challenge to attach thiolated DNA on AuNRs, because
the positively charged AuNRs readily aggregate in the presence of
negatively charged DNA. This article reports an mPEG-SH/Tween 20-assisted
method to load thiolated DNA on AuNRs in 1 h. Tween 20 and mPEG-SH
are used to synergistically displace CTAB on the surface of AuNRs
by repeated centrifugation and resuspension, and thiolated DNA are
attached to AuNRs in the presence of 1 M NaCl, 100 mM MgCl<sub>2</sub>, or 100 mM citrate. AuNRs with different sizes and aspect ratios
can be functionalized with DNA by this method. The number of DNA loaded
on each AuNR can be easily controlled by the concentrations of mPEG-SH
and Tween 20 or the ratio between DNA and AuNR. Functionalized AuNRs
were used for nanoparticle assembly and cancer cell imaging to confirm
that DNA anchored on the surface of AuNRs retains its hybridization
and molecular recognition capability. The new method is easy, rapid,
and robust for the preparation of DNA functionalized AuNRs for a variety
of applications such as cancer therapy, drug delivery, self-assembly,
and imaging
A Cell-Surface-Anchored Ratiometric Fluorescent Probe for Extracellular pH Sensing
Accurate sensing of the extracellular
pH is a very important yet challenging task in biological and clinical
applications. This paper describes the development of an amphiphilic
lipid–DNA molecule as a simple yet useful cell-surface-anchored
ratiometric fluorescent probe for extracellular pH sensing. The lipid–DNA
probe, which consists of a hydrophobic diacyllipid tail and a hydrophilic
DNA strand, is modified with two fluorescent dyes; one is pH-sensitive
as pH indicator and the other is pH-insensitive as an internal reference.
The lipid–DNA probe showed sensitive and reversible response
to pH change in the range of 6.0–8.0, which is suitable for
most extracellular studies. In addition, based on simple hydrophobic
interactions with the cell membrane, the lipid–DNA probe can
be easily anchored on the cell surface with negligible cytotoxicity,
excellent stability, and unique ratiometric readout, thus ensuring
its accurate sensing of extracellular pH. Finally, this lipid–DNA-based
ratiometric pH indicator was successfully used for extracellular pH
sensing of cells in 3D culture environment, demonstrating the potential
applications of the sensor in biological and medical studies
Integrating Target-Responsive Hydrogel with Pressuremeter Readout Enables Simple, Sensitive, User-Friendly, Quantitative Point-of-Care Testing
Point-of-care testing
(POCT) with the advantages of speed, simplicity,
and low cost, as well as no need for instrumentation, is critical
for the measurement of analytes in a variety of environments lacking
access to laboratory infrastructure. In the present study, a hydrogel
pressure-based assay for quantitative POCT was developed by integrating
a target-responsive hydrogel with pressuremeter readout. The target-responsive
hydrogels were constructed with DNA grafted linear polyacrylamide
and the cross-linking DNA for selective target recognition. The hydrogel
response to the target substance allows release of the preloaded Pt
nanoparticles, which have good stability and excellent catalytic ability
for decomposing H<sub>2</sub>O<sub>2</sub> to O<sub>2</sub>. Then,
the generated O<sub>2</sub> in a sealed environment leads to significant
pressure increase, which can be easily read out by a handheld pressuremeter.
Using this target-responsive hydrogel pressure-based assay, portable
and highly sensitive detection of cocaine, ochratoxin A, and lead
ion were achieved with excellent accuracy and selectivity. With the
advantages of portability, high sensitivity, and simple sample processing,
the hydrogel pressure-based assay shows great potential for quantitative
POCT of a broad range of targets in resource-limited settings
Design and Synthesis of Target-Responsive Aptamer-Cross-linked Hydrogel for Visual Quantitative Detection of Ochratoxin A
A target-responsive aptamer-cross-linked
hydrogel was designed and synthesized for portable and visual quantitative
detection of the toxin Ochratoxin A (OTA), which occurs in food and
beverages. The hydrogel network forms by hybridization between one
designed DNA strand containing the OTA aptamer and two complementary
DNA strands grafting on linear polyacrylamide chains. Upon the introduction
of OTA, the aptamer binds with OTA, leading to the dissociation of
the hydrogel, followed by release of the preloaded gold nanoparticles
(AuNPs), which can be observed by the naked eye. To enable sensitive
visual and quantitative detection, we encapsulated Au@Pt core–shell
nanoparticles (Au@PtNPs) in the hydrogel to generate quantitative
readout in a volumetric bar-chart chip (V-Chip). In the V-Chip, Au@PtNPs
catalyzes the oxidation of H<sub>2</sub>O<sub>2</sub> to generate
O<sub>2</sub>, which induces movement of an ink bar to a concentration-dependent
distance for visual quantitative readout. Furthermore, to improve
the detection limit in complex real samples, we introduced an immunoaffinity
column (IAC) of OTA to enrich OTA from beer. After the enrichment,
as low as 1.27 nM (0.51 ppb) OTA can be detected by the V-Chip, which
satisfies the test requirement (2.0 ppb) by the European Commission.
The integration of a target-responsive hydrogel with portable enrichment
by IAC, as well as signal amplification and quantitative readout by
a simple microfluidic device, offers a new method for portable detection
of food safety hazard toxin OTA