Nucleic acid biomarkers have been widely used to detect various viral-associated diseases, including the recent pandemic COVID-19. The CRISPR-Cas-based trans-activating phenomenon has shown excellent potential for developing sensitive and selective detection of nucleic acids. However, the nucleic acid amplification steps are typically required when sensitive and selective monitoring of the target nucleic acid is needed. To overcome the aforementioned challenges, we developed a CRISPR-Cas12a-based nucleic acid amplification-free biosensor by a surface-enhanced Raman spectroscopy (SERS)-assisted ultrasensitive detection system. We integrated the activated CRISPR-Cas12a by viral DNA with a Raman-sensitive system composed of ssDNAimmobilized Raman probe-functionalized Au nanoparticles (RAuNPs) on the graphene oxide (GO)/triangle Au nanoflower array. Using this CRISPR-based Raman-sensitive system improved the detection sensitivity of the multiviral DNAs such as hepatitis B virus (HBV), human papillomavirus 16 (HPV-16), and HPV-18 with an extremely low detection limit and vast detection range from 1 aM to 100 pM without the amplification steps. We suggest that this ultrasensitive amplification-free detection system for nucleic acids can be widely applied to the precise and early diagnosis of viral infections, cancers, and several genetic diseases.

In this research, we developed electrochemical biosensor which was composed of hemoglobin (Hb)-DNA conjugate on nanoporous gold thin film (NPGF) for hydrogen peroxide (H2O2) detection. For the first time, Hb and DNA was conjugated as a sensing platform for uniform orientation of Hb on electrode. The newly developed Hb-DNA conjugate was designed to prevent Hb from aggregation on electrode. DNA hybridization of Hb-DNA conjugate and complementary DNA (cDNA) on NPGF electrode induced uniformly assembled biosensor. Furthermore, NPGF electrode fabrication method was introduced to the increment of the surface area. To confirm the conjugation of Hb-DNA conjugate, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ultraviolet–visible spectroscopy (UV–VIS) were used. Formation of the NPGF electrode was verified by scanning electron microscope (SEM). Atomic force microscopy (AFM) was operated for the confirmation of Hb-DNA immobilization on electrode. The electrochemical property of fabricated electrode was investigated by cyclic voltammetry (CV). Also, H2O2 sensing performance of fabricated electrode was investigated by amperometric i-t curve technique. This sensor showed a wide linear range from 0.00025 to 5.00 mM and a correlation coefficient of R2 = 0.9986. The detection limit was 250 nM. Proposed biosensor can be utilized as a sensing platform for development of biosensor.Electronic supplementary materialThe online version of this article (10.1186/s40580-018-0172-z) contains supplementary material, which is available to authorized users.

Biosensors are very important for detecting target molecules with high accuracy, selectivity, and signal-to-noise ratio. Biosensors developed using biomolecules such as enzymes or nucleic acids which were used as the probes for detecting the target molecules were studied widely due to their advantages. For example, enzymes can react with certain molecules rapidly and selectively, and nucleic acids can bind to their complementary sequences delicately in nanoscale. In addition, biomolecules can be immobilized and conjugated with other materials by surface modification through the recombination or introduction of chemical linkers. However, these biosensors have some essential limitations because of instability and low signal strength derived from the detector biomolecules. Functional nanomaterials offer a solution to overcome these limitations of biomolecules by hybridization with or replacing the biomolecules. Functional nanomaterials can give advantages for developing biosensors including the increment of electrochemical signals, retention of activity of biomolecules for a long-term period, and extension of investigating tools by using its unique plasmonic and optical properties. Up to now, various nanomaterials were synthesized and reported, from widely used gold nanoparticles to novel nanomaterials that are either carbon-based or transition-metal dichalcogenide (TMD)-based. These nanomaterials were utilized either by themselves or by hybridization with other nanomaterials to develop highly sensitive biosensors. In this review, highly sensitive biosensors developed from excellent novel nanomaterials are discussed through a selective overview of recently reported researches. We also suggest creative breakthroughs for the development of next-generation biosensors using the novel nanomaterials for detecting harmful target molecules with high sensitivity.