Circulating tumor cells (CTCs) contain molecular information on the primary tumor and can be used for predictive cancer diagnostics. Capturing rare live CTCs and their quantification in whole blood remain technically challenging. Here we report an aptamer-trigger clamped hybridization chain reaction (atcHCR) method for in situ identification and subsequent cloaking/decloaking of CTCs by porous DNA hydrogels. These decloaked CTCs were then used for live cell analysis. In our design, a DNA staple strand with aptamer-toehold biblocks specifically recognizes epithelial cell adhesion molecule (EpCAM) on the CTC surface that triggers subsequent atcHCR via toehold-initiated branch migration. Porous DNA hydrogel based-cloaking of single/cluster of CTCs allows capturing of living CTCs directly with minimal cell damage. The ability to identify a low number of CTCs in whole blood by DNA hydrogel cloaking would allow high sensitivity and specificity for diagnosis in clinically relevant settings. More significantly, decloaking of CTCs using controlled and defined chemical stimuli can release living CTCs without damages for subsequent culture and live cell analysis. We expect this liquid biopsy tool to open new powerful and effective routes for cancer diagnostics and therapeutics.Keywords: Circulating tumor cell; CTC capture; CTC release; DNA hydrogel; live cell analysis;

We recognize the stochastic collisions of dopamine contained phospholipid vesicle on carbon fiber nanoelectrode, extending the observation of discrete collision events on nanoelectrode to biologically relevant analytes. To decrease noise interference to the technique, the dimensions of nanoelectrode was systematically investigated and optimized. Scanning electron microscopy (SEM) further supported the comparable sizes of nanoelectrode and vesicles (~100 nm in diameter). Vesicles collision and rupture on the surface of nanoelectrode led to the dopamine release from vesicles, which could be electrochemically oxidized to dopamine-o-quinone and detected via voltammetry. The comparable size of the nanoelectrode with vesicles and fast voltammetry allowed differentiation of single collision events from the current magnitudes and peak widths in the electrochemical collision experiments, which shows the efficacy of the method to characterize vesicle samples. This work provides a foundation upon which quantitative sensor technology might be built for the detection of dopamine contained vesicles with high spatial and temporal resolution.

To better understand cell behaviors on substrates, the precise control of density and orientation of cell-specific ligands remains a great challenge. In this study, we established an easy-to-use approach to manipulate the adhesion and patterning of mammalian cells on gold substrates. We prepared DNA self-assembled monolayers (DNA-SAMs) on gold substrates and found that the sequence-specific orientation of DNA-SAMs played an important role in modulating cell adhesion. We also found that the DNA-SAMs on gold substrates could be used as a potentially universal cell culture substrate, which showed properties similar to cationic polymers (e.g. poly-L lysine, PLL) substrates. Furthermore, we could manipulate cell adhesion by tuning the length of poly adenine (polyA) in the DNA sequence. We also prepared a DNA aptamer-based SAM to regulate cell adhesion by exploiting stimuli-responsive conformational change of the aptamer. By using the well-established DNA spotting technology, we patterned cells on DNA-SAMs to form a spot matrix and four English letters “CELL”. Our findings suggest that DNA-SAMs on gold substrates are potentially useful for making smart surfaces for cell studies, thus introducing a new platform for cell/tissue engineering research.

The fixed dynamic range of traditional biosensors limits their utility in several real applications. For example, viral load monitoring requires the dynamic range spans several orders of magnitude; whereas, monitoring of drugs requires extremely narrow dynamic range. To overcome this limitation, here, we devised tunable biosensing interface using allosteric DNA tetrahedral bioprobes to tune the dynamic range of DNA biosensors. Our strategy takes the advantage of the readily and flexible structure design and predictable geometric reconfiguration of DNA nanotechnology. We reconfigured the DNA tetrahedral bioprobes by inserting the effector sequence into the DNA tetrahedron, through which, the binding affinity of DNA tetrahedral bioprobes can be tuned. As a result, the detection limit of DNA biosensors can be programmably regulated. The dynamic range of DNA biosensors can be tuned (narrowed or extended) for up to 100-fold. Using the regulation of binding affinity, we realized the capture and release of biomolecules by tuning the binding behavior of DNA tetrahedral bioprobes.

•A smartphone interfaced electrochemical chip device for the on-site gender verification was developed.•The two enzymes joint detection ensured the clear discrimination of male and female group.•This device retained well when we used the device in serum and serum stains.•The whole detection can be completed within 20 min.•The portability of smartphone based device is well suitable in the applications such as on-site analysis.On-site detection of biomarkers in biofluids found at the crime scene is critically important forensic analysis. However, it remains difficult due to the lack of portable, fast and cheap on-site analytical devices. Traditional methods including polymerase chain reaction (PCR) and electrophoresis requires complicated instrumentation and critical environment. Optical analytical methods can be affected by intrinsic adsorption of complicated sample such as serum. In response, we developed a smartphone-interfaced electrochemical chip device for on-site gender verification. The detection is based on the known difference of biomarkers (creatine kinase (CK) and alanine transaminase (ALT)) between male and female groups. Enzyme cascade reaction converted the enzyme level in biofluids to the consumption of NADH, which can be electrochemically detected by our designed electrochemical chip. Our device retained the capability of gender verification when used in serum and serum stains. The detection can be completed in 20 min. Gender verification of real samples (39 serum samples) demonstrated excellent sensitivity and specificity of this smartphone based device.

•A high sensitive electrochemical aptasensor was fabricated.•DNA aptamer was modified onto gold nanoparticles to form a nanoprobe.•Terminal deoxynucleotidyl transferase (TdT) was employed to amplify the signals.•A detection limit of 5 fM target was achieved.Herein, an aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) strategy for electrochemical aptasensor (E-aptasensor) is developed for analysis of cancer biomarker carcino-embryonic antigen (CEA). A pair of DNA aptamers is employed which can be specifically bond with CEA simultaneously. One of the aptamer is thiolated at 3′-terminal and immobilized onto the gold electrode as a capture probe, while the other one has a thiol group at its 5′-terminal and is modified onto the gold nanoparticles surface to form a nanoprobe. In the present of target, the two aptamers can “sandwich” the target, thus the nanoprobe is attached to the electrode. Then terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the incorporation of biotin labeled dNTPs into the 3′–OH terminals of the DNA aptamer on the nanoprobe. The as-generated long DNA oligo tentacles allow specific binding of numerous avidin modified horseradish peroxidase (Av-HRP), resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. Taking advantage of the enzyme based nucleic acid amplification and nanoprobe, this strategy is demonstrated to possess the outstanding amplification efficiency.

Enzyme complexes are assembled at the two-dimensional lipid membrane or prearranged on three-dimensional scaffolding proteins to regulate their catalytic activity in cells. Inspired by nature, we have developed gold nanoparticle-based spherical DNAzymes (SNAzymes) with programmably engineered activities by exploiting poly-adenine (polyA)–Au interactions. In a SNAzyme, AuNPs serve as the metal core, which is decorated with a functional shell of DNAzymes. Conventional thiolated DNAzyme-based assembly leads to disordered structures with suppressed activity. In contrast, by using an anchoring block of polyA tails, we find that the activity of SNAzymes can be programmably regulated. By using a polyA30 tail, SNAzymes demonstrated remarkably enhanced binding affinity compared to the thiolated DNAzyme-based assembly (∼75-fold) or individual DNAzymes in the solution phase (∼10-fold). More significantly, this increased affinity is directly translated to the sensitivity improvement in the SNAzyme-based lead sensor. Hence, this design of SNAzymes may provide new opportunities for developing biosensors and bioimaging probes for theranostic applications.

A novel biosensor platform was developed for detection of microRNAs (miRNAs) based on graphene quantum dots (GQDs) and pyrene-functionalized molecular beacon probes (py-MBs). Pyrene was introduced to trigger specifically fluorescence resonance energy transfer (FRET) between GQDs and fluorescent dyes labeled on py-MBs, and the unique fluorescent intensity change produced a novel signal for detection of the target. The platform realized detection of miRNAs in a wide range from 0.1 nM to 200 nM with great discrimination abilities, as well as multidetection of different kinds of miRNAs, which paved a brand new way for miRNA detection based on GQDs.Keywords: biosensor; fluorescence resonance energy transfer; graphene quantum dots; microRNA; pyrene

DNA-decorated metal nanoparticles have found numerous applications, most of which rely on thiolated DNA (SH-DNA)-modified gold nanoparticles (AuNPs). Whereas silver nanoparticles (AgNPs) are known to have stronger plasmonic properties than AuNPs, modification of AgNPs with SH-DNA is technically challenging, partially due to the instability of Ag–S bonding. Here we demonstrate a facile approach to self-assemble unmodified DNA on AgNPs by exploiting intrinsic silver–cytosine (Ag–C) coordination. The strong Ag–C coordination allows for the ready formation of DNA-AgNP conjugates, which show favorable stability under conditions of high ionic strength and high temperature. These nanoconjugates possess much higher efficient molecular recognition capability and faster hybridization kinetics than thiolated DNA-modified AgNPs. More importantly, we could programmably tune the DNA density on AgNPs with the regulation of silver–cytosine coordination numbers, which in turn modulated their hybridizability. We further demonstrated that these DNA-AgNP conjugates could serve as excellent building blocks for assembling silver and hybrid silver–gold nanostructures with superior plasmonic properties.Keywords: coordination; plasmonic; programmable assembly; silver nanoparticle; umodified DNA;

Electrochemical DNA (E-DNA) sensors have been greatly developed and play an important role in early diagnosis of different diseases. To determine the extremely low abundance of DNA biomarkers in clinical samples, scientists are making unremitting efforts toward achieving highly sensitive and selective E-DNA sensors. Here, a novel E-DNA sensor was developed taking advantage of the signal amplification efficiency of nanoprobe-initiated enzymatic polymerization (NIEP). In the NIEP based E-DNA sensor, the capture probe DNA was thiolated at its 3′-terminal to be immobilized onto gold electrode, and the nanoprobe was fabricated by 5′-thiol-terminated signal probe DNA conjugated gold nanoparticles (AuNPs). Both of the probes could simultaneously hybridize with the target DNA to form a “sandwich” structure followed by the terminal deoxynucleotidyl transferase (TdT)-catalyzed elongation of the free 3′-terminal of DNA on the nanoprobe. During the DNA elongation, biotin labels were incorporated into the NIEP-generated long single-stranded DNA (ssDNA) tentacles, leading to specific binding of avidin modified horseradish peroxidase (Av-HRP). Since there are hundreds of DNA probes on the nanoprobe, one hybridization event would generate hundreds of long ssDNA tentacles, resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. By employing nanoprobe and TdT, it is demonstrated that the NIEP amplified E-DNA sensor has a detection limit of 10 fM and excellent differentiation ability for even single-base mismatch.Keywords: electrochemical DNA (E-DNA) sensors; nanoprobe; signal amplification; terminal deoxynucleotidyl transferase (TdT)

•We introduced a novel strategy of E-DNA sensor based on double DNA tetrahedral nanostructures.•We proposed the DNA tetrahedral nanostructures as signal amplification reporters.•Our sensor was able to detect 1 fM DNA targets and obtained a large dynamic range.Electrochemical DNA (E-DNA) sensor is an important tool for detecting DNA biomarker. In this work, we have demonstrated a novel strategy of E-DNA sensor based on DNA tetrahedral nanostructures for the sensitive detection of target DNA. In our design, thiol and biotin modified DNA tetrahedral nanostructures were used as capture and report probes respectively. The biotin-tagged three dimensional DNA tetrahedral nanostructures were employed for efficient signal amplification by capturing multiple catalytic enzymes. Such improved E-DNA sensor can sensitively detect DNA target as low as 1 fM with excellent differentiation ability for even single mismatch. And a mean recovery rate of 90.57% in DNA solution extracted from human serum was obtained. We have also compared this new method of attaching catalytic enzymes with the other two typical methods: One is through biotinylated single-stranded DNA (SSDNA) and the other is through gold nanoparticles (GNPs). Results indicated that the RTSPs-based enzyme amplification system showed much better performance than the other two systems.

•The binding of ATP triggered the collapse of the DNA superstructures.•The detection time was significantly decreased to 10 min.•The results can be recognized by naked eyes.The detection of small molecules depends heavily on complicated GC–MS (Gas chromatography–mass spectrometry), HPLC (High-performance liquid chromatography) and some other complicated instruments that are not suitable for point of care detection. Here, we have demonstrated a fast (in 10 min), simple (instrument-free) and effective detection platform for small molecule-ATP. In our design, we engineered the hybridization region of aptamer and assembled it into a superstructure to avoid the exposed flexible ends. The binding of ATP triggered the collapse of the superstructures to produce single stranded DNA that can obviously tune the plasmonic coupling of unmodified gold nanoparticles (AuNPs). Compared to detection platforms based on fully hybridized aptamer double helix, the detection time was significantly decreased to 10 min. The resulting color change can be recognized by naked eyes. Our detection is highly specific and selective. Furthermore, a logic gate with multiplexed detection capability for ATP and DNA were demonstrated.

Nanomechanical switching of functional three-dimensional (3D) DNA nanostructures is crucial for nanobiotechnological applications such as nanorobotics or self-regulating sensor and actuator devices. Here, DNA tetrahedral nanostructures self-assembled onto gold electrodes were shown to undergo the electronically addressable nanoswitching due to their mechanical reconfiguration upon external chemical stimuli. That enables construction of robust surface-tethered electronic nanodevices based on 3D DNA tetrahedra. One edge of the tetrahedron contained a partially self-complementary region with a stem-loop hairpin structure, reconfigurable upon hybridization to a complementary DNA (stimulus DNA) sequence. A non-intercalative ferrocene (Fc) redox label was attached to the reconfigurable tetrahedron edge in such a way that reconfiguration of this edge changed the distance between the electrode and Fc.Keywords: 3D nanostructures; DNA nanotechnology; DNA tetrahedron; electromechanical devices; nanomechanical switching; self-assembly;

The high occurrence of prostate cancer in men makes the prostate-specific antigen (PSA) screening test really important. More importantly, the recurrence rate after radical prostatectomy is high, whereas the traditional PSA immunoassay does not possess the sufficient high sensitivity for post-treatment PSA detection. In these assays, uncontrolled and random orientation of capture antibodies on the surface largely reduces their activity. Here, by exploiting the rapidly emerging DNA nanotechnology, we developed a DNA nanostructure based scaffold to precisely control the assembly of antibody monolayer. We demonstrated that the detection sensitivity was critically dependent on the nanoscale-spacing (nanospacing) of immobilized antibodies. In addition to the controlled assembly, we further amplified the sensing signal by using the gold nanoparticles, resulting in extremely high sensitivity and a low detection limit of 1 pg/mL. To test the real-world applicability of our nanoengineered electrochemical sensor, we evaluated the performance with 11 patients’ serum samples and obtained consistent results with the “gold-standard” assays.

There remains a great challenge in the sensitive detection of microRNA because of the short length and low abundance of microRNAs in cells. Here, we have demonstrated an ultrasensitive detection platform for microRNA by combining the tetrahedral DNA nanostructure probes and hybridization chain reaction (HCR) amplification. The detection limits for DNA and microRNA are 100 aM and 10 aM (corresponding to 600 microRNAs in a 100 μL sample), respectively. Compared to the widely used supersandwich amplification, the detection limits are improved by 3 orders of magnitude. The uncontrolled surface immobilization and consumption of target molecules that limit the amplification efficiency of supersandwich are eliminated in our platform. Taking advantage of DNA nanotechnology, we employed three-dimensional tetrahedral DNA nanostructure as the scaffold to immobilize DNA recognition probes to increase the reactivity and accessibility, while DNA nanowire tentacles are used for efficient signal amplification by capturing multiple catalytic enzymes in a highly ordered way. The synergetic effect of DNA tetrahedron and nanowire tentacles have proven to greatly improve sensitivity for both DNA and microRNA detection.

Sensitive detection of cancer cells plays a critically important role in the early detection of cancer and cancer metastasis. However, because circulating tumor cells are extremely rare in peripheral blood, the detection of cancer cells with high analytical sensitivity and specificity remains challenging. Here, we have demonstrated a simple, sensitive and specific detection of cancer cells with the detection sensitivity of four cancer cells, which is lower than the cutoff value with respect to correlation with survival outcomes as well as predictive of metastatic disease in clinical diagnostics. We re-engineered the hybridization chain reaction (HCR) to multibranched HCR (mHCR) that can produce long products with multiple biotins for signal amplification and multiple branched arms for multivalent binding. The capturing gold surface is modified with DNA tetrahedral probes, which provide superior hybridization conditions for the multivalent binding. The synergetic effect of mHCR amplification and multivalent binding lead to the high sensitivity of our detection platform.

To better understand cell behaviors on substrates, the precise control of density and orientation of cell-specific ligands remains a great challenge. In this study, we established an easy-to-use approach to manipulate the adhesion and patterning of mammalian cells on gold substrates. We prepared DNA self-assembled monolayers (DNA-SAMs) on gold substrates and found that the sequence-specific orientation of DNA-SAMs played an important role in modulating cell adhesion. We also found that the DNA-SAMs on gold substrates could be used as a potentially universal cell culture substrate, which showed properties similar to cationic polymers (e.g. poly-L lysine, PLL) substrates. Furthermore, we could manipulate cell adhesion by tuning the length of poly adenine (polyA) in the DNA sequence. We also prepared a DNA aptamer-based SAM to regulate cell adhesion by exploiting stimuli-responsive conformational change of the aptamer. By using the well-established DNA spotting technology, we patterned cells on DNA-SAMs to form a spot matrix and four English letters “CELL”. Our findings suggest that DNA-SAMs on gold substrates are potentially useful for making smart surfaces for cell studies, thus introducing a new platform for cell/tissue engineering research.