Active Polymerase on Metal-Organic Frameworks: Space-Confined Amplification for the Extraction-Free Detection of...

The presence of numerous inhibitors and genomic DNA in blood makes their direct use for analysis of clinical pathogens by nucleic acid amplification techniques difficult. Herein, an in situ biomimetic immobilized enzyme system featuring nucleic acid amplification within nanoconfined spaces and self-cleaning properties was constructed for rapid and direct absolute quantification of bacterial bloodstream infections in clinical blood samples without blood culture and extraction steps. The metal-organic framework (MOF) acts as an enzyme-protective carrier, providing 3D nanoconfined spaces, shielding enzymes from harsh environments and permitting inhibition-free nucleic acid analysis directly in blood. Simultaneously, the nanoconfined environments with nanoporous structures possess adsorption, restriction, separation, release, and self-cleaning abilities. When PCR is performed for pathogen detection without any extraction of whole blood, the reagents will be absorbed and released on demand by the surrounding nanostructures during or after amplification, resulting in a faster amplification rate, robust anti-inhibition, enhanced signal readout, and specific amplification without primer-dimer. Quantification of bacteria, such as Escherichia coli and Staphylococcus aureus, in whole blood was achieved with a detection limit as low as 10 CFU/mL, representing a sensitivity improvement of two or three orders of magnitude. Moreover, the assay was validated by using 15 clinical blood samples (100% sensitivity and specificity) and dramatically shortened the sample-to-result time from over 48 h to approximately 1 h. This technology holds great potential in the detection of various blood-related diseases for clinical laboratory diagnostics, especially for bacteria and viruses, administering timely, optimal treatment and improving the quality of health care for humans.