Construction of a monoclonal molecular imprinted sensor with high affinity for specific recognition of influenza a virus subtype.

Although molecular imprinting technology has been widely used in the construction of virus sensors, it is still a great challenge to identify subtypes viruses specifically because of their high similarity in morphology, size and structure. Here, a monoclonal molecular imprinted polymers (MIPs) sensor for recognition of H5N1 is constructed to permit the accurate distinguishing of H5N1 from other influenza A virus (IAV) subtypes. Firstly, H5N1 are immobilized on magnetic microspheres to produce H5N1-MagNPs, then the high affinity nanogel H5N1-MIPs is prepared by solid phase imprinting technique. When H5N1-MIPs is combined with MagNP-H5N1, different concentrations of H5N1 are added for competitive substitution. The quantitative detection of H5N1 is realized by the change of fluorescence intensity of supernatant. As expected, the constructed sensor shows satisfactory selectivity, and can identify the target virus from highly similar IAV subtypes, such as H1N1, H7N9 and H9N2. The sensor was highly sensitive, with a detection limit of 0.58 fM, and a selectivity factor that is comparable to that of other small MIPs sensors is achieved. In addition, the proposed sensor is cheap, with a cost of only RMB 0.08 yuan. The proposed monoclonal sensor provides a new method for the specific recognition of designated virus subtype, which is expected to be used for large-scale screening and accurate treatment of infected people.

A point-to-point "cap" strategy to construct a highly selective dual-function molecularly-imprinted sensor for the simultaneous detection of HAV and HBV.

As an artificial biomimetic receptor, molecularly-imprinted polymer (MIP) has been widely used for the separation, enrichment and detection of various substances. However, due to the complexity of virus structure, huge volume and the existence of highly similar viruses, MIP shows unsatisfactory selectivity in virus detection. To overcome these issues, two kinds of virus nanoMIPs, just like a "cap", were synthesized by a solid-phase imprinting nanogel technique. The "cap" had no inner core and was much smaller than that of a conventional MIP, which was more favorable for mass transfer. Moreover, each "cap" could only combine with one target virus, which avoided the interference between large-volume virus molecules effectively. The two synthesized "caps" were mixed to construct a bifunctional MIP virus sensor for the simultaneous detection of Hepatitis A virus (HAV) and Hepatitis B virus (HBV). As expected, the selectivity factor (SF) for HBV detection reached 13.7, which was much higher than the reported virus MIP sensors (SF: 3-6), which was comparable to that of small molecular imprinting sensors. In addition, the high sensitivity toward HBV was 34.3 fM, and that of HAV was 27.1 pM. This method provides an idea for preparing high-selectivity biomacro-MIPs, as well as a method for the simultaneous detection of similar viruses with high sensitivity and selectivity. The recovery experiment of spiked serum showed that this method also has great practical application prospects.

Non-autofluorescence Detection of H5N1 Virus Using Photochemical Aptamer Sensors Based on Persistent Luminescent Nanoparticles.

Fluorescence sensing is limited in practical applications owing to multiple autofluorescent substances in complex biological samples such as serum. In this paper, the luminescence decay effect of persistent luminescent nanoparticles (PLNPs) was used to avoid the interference of autofluorescence in complex biological samples, and a non-autofluorescence molecularly imprinted polymer aptamer sensor (MIP-aptasensor) was designed to detect H5N1 virus. The proposed MIP-aptasensor consists of a magnetic MIP and aptamer-functionalized persistent luminescent nanoparticle Zn2GeO4:Mn2+-H5N1 aptamer (ZGO-H5N1 Apt). Upon simultaneous recognition of H5N1 virus, strong persistent luminescent signal changes were produced. Using the unique luminescent characteristics of PLNPs and the high selectivity of imprinted polymers and aptamers, the designed MIP-aptasensor effectively eliminates the autofluorescence background interference of serum samples and realizes the non-autofluorescence detection of H5N1 virus with high sensitivity (a limit of detection of 0.0128 HAU mL-1, 1.16 fM) and selectivity (the imprinting factor for the target H5N1 virus was 6.72). This tool provides a strategy for the design of sensors and their application in complex biological samples.