We review the recent advances in quantum dot-based biosensors and focus on quantum dot-based fluorescent, bioluminescent, chemiluminescent, and photoelectrochemical biosensors.

Single-molecule detection provides a simple and ultrasensitive platform to quantify target molecules by simply counting the individual fluorescence signals. Quantum dots (QDs) are novel semiconductor nanocrystals with distinct characteristics of high brightness, large Stokes shift and broad absorption spectra, high molar extinction coefficients, high quantum yield, good photostability and long fluorescence lifetime. The combination of single-molecule detection with QDs enables the development of single QD-based nanosensors with extremely high sensitivity. Single QD-based nanosensors may be divided into two categories based on single QD burst coincidence detection and single QD-fluorescence resonance energy transfer (FRET) detection, and have significant advantages of high signal-to-noise ratio, high sensitivity, rapidity, and low sample consumption. The single QD-based nanosensors have the capability of directly detecting low-abundance species without the need for nucleic acid amplification, and may elucidate a variety of biological and biochemical phenomena in real time using single QD tracking. In this review, we summarize the recent advances in single QD-based nanosensors and their applications for sensitive detection of DNAs, microRNAs, proteins, enzymes, small molecules and viruses. We highlight the challenges and future direction of single QD-based nanosensors as well.

Specific and sensitive detection of DNA MTase activity can be achieved by a single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification approach.

The orbitofrontal cortex (OFC) is thought to link stimuli and actions with anticipated outcomes in order to sustain flexible behavior in an ever-changing environment. How it retains these associations to guide future behavior is less well-defined. Here we focused on one subregion of this heterogeneous structure, the ventrolateral OFC (VLO). CaMKII-driven inhibitory Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) were infused and subsequently activated by their ligand Clozapine-N-oxide (CNO) in conjunction with fear extinction training (a form of aversive conditioning) and response-outcome conditioning (a form of appetitive conditioning). Gi-DREADD-mediated inactivation of the VLO during extinction conditioning interfered with fear extinction memory, resulting in sustained freezing when mice were later tested drug-free. Similarly, Gi-DREADD-mediated inactivation in conjunction with response-outcome conditioning caused a later decay in goal-directed responding-that is, mice were unable to select actions based on the likelihood that they would be rewarded in a sustainable manner. By contrast, inhibitory Gi-DREADDs in the basolateral amygdala (BLA) impaired the acquisition of both conditioned fear extinction and response-outcome conditioning, as expected based on prior studies using other inactivation techniques. Meanwhile, DREADD-mediated inhibition of the dorsolateral striatum enhanced response-outcome conditioning, also in line with prior reports. Together, our findings suggest that learning-related neuroplasticity in the VLO may be necessary for memory retention in both appetitive and aversive domains.

DNA glycosylases are responsible for recognition and excision of the damaged bases in the base excision repair pathway, and all mammals express multiple DNA glycosylases to maintain genome stability. However, simultaneous detection of multiple DNA glycosylase still remains a great challenge. Here, we develop a rapid and sensitive fluorescent method for simultaneous detection of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycolase (UDG) using exonuclease-assisted recycling signal amplification in combination with fluorescent bases 2-aminopurine (2-AP) and pyrrolo-dC (P-dC) as the fluorophores. We design a bifunctional DNA probe modified with one 8-oxoG and five uracil bases, which can hybridize with the trigger probes to form a sandwiched DNA substrate for hOGG1 and UDG. In addition, we design 2-AP and P-dC signal probes as the hairpin structures with 2-AP and P-dC in the stems. The presence of hOGG1 and UDG may initiate the signal amplification process by the recycling lambda exonuclease digestion and generates distinct fluorescence signals, with 2-AP indicating the presence of hOGG1 and P-dC indicating the presence of UDG. This method can simultaneously detect multiple DNA glycosylases with the detection limits of 0.0035 U/mL for hOGG1 and 0.0025 U/mL for UDG, and it can even measure DNA glycosylases at the single-cell level. Moreover, this method can be applied for the measurement of enzyme kinetic parameters and the screening of DNA glycosylase inhibitors, holding great potential for further applications in biomedical research and clinical diagnosis.

We demonstrate for the first time the development of a mimic peroxidase- and bismuth sulfide (BiS) nanorod-based photoelectrochemical (PEC) biosensor for signal-on detection of polynucleotide kinase (PNK) on the basis of manganese-based mimic enzyme (MnME) catalytic precipitation. We use the hybrid film which consists of BiS nanorods and Au nanoparticles (AuNPs) as the immobilization matrix of capture probe. The capture probe on the modified electrode can specifically hybridize with the MnME@AuNPs-labeled signal probe to form the double-stranded DNA (dsDNA), generating a PEC biosensor. In the absence of PNK, MnME may stimulate the mimic enzyme catalytic precipitation onto the electrode surface, blocking the interfacial electron transfer and eventually leading to a low PEC signal. While in the presence of PNK, the dsDNA is phosphorylated and subsequently cleaved by lambda exonuclease to release the MnME@AuNPs conjugates from the electrode, leading to the decrease of catalytic precipitation on the surface of electrode and consequently the production of a high PEC signal. Notably, the MnME can be easily synthesized and possesses higher catalytic activity than the manganese-based mimic enzyme. This signal-on PEC biosensor exhibits high sensitivity with a detection limit of 1.27 × 10 U mL and an extrembly large dynamic range of 5 orders of magnitude. Moreover, it can be applied for the screening of PNK inhibitors and accurate quantification of PNK activity in cancer cell extracts.

Integration of single-molecule detection with rolling circle amplification-driven encoding of different fluorescent molecules enables simultaneous detection of multiple DNA repair enzymes.

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