Nanodiamond Diagnostics—Improving Sensitivity by Spin Manipulation

The room-temperate spin properties of nitrogen-vacancy centres in fluorescent nanodiamond particles make them promising candidates for biosensing. Along with their high brightness, stability and low-cost, the selective manipulation of their fluorescence emission allows separation of their signal from background autofluorescence, which typically limits sensitivity.<br/>There is an urgent need for low-cost, rapid biosensors for early detection of diseases. For example, there are 38 million people living with HIV worldwide, of whom 7.1 million are unaware of their HIV status¹. Lateral flow tests meet many of the REASSURED diagnostic criteria², allowing multi-step assays with little user input. However, the most commonly used gold nanoparticle-based lateral flow tests often lack the required sensitivity for accurate early detection.<br/>We applied fluorescent nanodiamonds containing negative nitrogen-vacancy centres as labels on lateral flow³ to improve diagnostic analytical sensitivity. Nanodiamonds bind to the test line in the presence of the target analyte, after which we used fluorescence imaging to read results. To enhance signal-to-background ratio, and therefore sensitivity, we applied a continuous-wave microwave-frequency electromagnetic field to manipulate nanodiamond emission during imaging. This drives the electron spin population to the m_s=±1 state, reducing the photoluminescent intensity by increasing the probability of decay into a metastable ‘dark’ state. Amplitude-modulating this microwave field, and therefore the modulating fluorescence intensity at a fixed frequency, allows the use of frequency-domain analysis (computational lock-in) to separate the nanodiamond signal from the non-modulated background autofluorescence, improving sensitivity.<br/>This gave a detection limit on lateral flow of 820 zM with an idealised biotin-avidin model, just 27 particles in a 55 µL sample: an improvement of 10⁵-fold over gold nanoparticles. This includes a 100-fold improvement using microwave modulation over conventional fluorescence imaging. Extending to an amplicon detection assay gave a 3.7 fM detection limit, 7,500-fold lower than gold nanoparticles. Applying this assay to the detection of HIV RNA led to single-copy detection with the addition of a short 10-minute isothermal amplification step.<br/>Nanodiamonds are applicable to numerous diagnostic formats and targets, with the potential to dramatically improve sensitivity of low-cost diagnostics.<br/>(1) UNAIDS. Global HIV & AIDS statistics — 2020 fact sheet https://www.unaids.org/en/resources/fact-sheet (accessed Jan 7, 2021).<br/>(2) Land, K. J. et al. REASSURED Diagnostics to Inform Disease Control Strategies, Strengthen Health Systems and Improve Patient Outcomes. <i>Nat. Microbiol.</i> <b>2019</b>, <i>4</i> (1), 46–54. https://doi.org/10.1038/s41564-018-0295-3.<br/>(3) Miller, B. S. et al. Spin-Enhanced Nanodiamond Biosensing for Ultrasensitive Diagnostics. <i>Nature</i> <b>2020</b>, <i>587</i> (7835), 588–593. https://doi.org/10.1038/s41586-020-2917-1.