Early and accessible disease diagnostics, particularly for cancers, necessitate highly sensitive and label-free methods for detecting biomarkers such as microRNAs (miRNAs). Current methods often rely on fluorescence labeling and amplification, which are labor-intensive and can introduce artifacts. Field-effect transistor (FET) biosensors offer a label-free approach by measuring the intrinsic charge of biomolecules. However, these sensors suffer from a significant limitation: Debye screening. The negatively charged phosphate backbone of nucleic acids attracts counterions in solution, forming an ionic cloud that screens the biomolecule's charge within a Debye length (typically less than 1 nm). This significantly reduces the sensitivity of FET-based detection. To overcome this challenge, this research explores an alternative approach based on quantum sensing, using the unique properties of nitrogen-vacancy (NV) centers in diamond. NV centers are atomic-scale defects in the diamond lattice that possess exceptional sensitivity for detecting electric and magnetic fields. Their application in sensing proteins, nuclear magnetic resonance (NMR), and electron spin resonance (ESR) has been well-established. Previous research has demonstrated their ability to detect ESR spectra of DNA labeled with spin labels, sense viruses like SARS-CoV-2 and HIV, showcasing their versatility and sensitivity for biomedical applications. miRNAs, short non-coding RNA molecules (20-22 nucleotides), are promising biomarkers for various cancers, neurodegenerative, and autoimmune diseases. Traditional methods for miRNA detection (Northern blotting, quantitative PCR, microarrays) suffer from limitations in sensitivity, require labor-intensive steps, and often necessitate specialized equipment. This paper proposes to use NV centers as a novel quantum sensing platform for label-free miRNA detection, leveraging the magnetic noise generated by the interaction between miRNAs and their paramagnetic counterions to overcome the limitations of Debye screening.

The challenge of detecting biomolecules at low concentrations without labels has driven innovation in biosensor technology. Significant work has focused on field-effect transistors, utilizing the charge of the biomolecule to induce a measurable change in current. However, the Debye screening effect, where counterions shield the biomolecule's charge, has been a major hurdle. Several studies have aimed to overcome this limitation by modifying sensor surfaces or using novel materials to enhance sensitivity. Meanwhile, quantum sensing using NV centers in diamond has emerged as a powerful technique for detecting various physical quantities with high precision. NV centers' sensitivity to electric and magnetic fields has been exploited for diverse applications, including sensing magnetic fields at the nanoscale, detecting NMR and ESR signals from single molecules, and biosensing applications. Studies have shown that NV centers can detect single proteins, DNA, and viruses with remarkable sensitivity, highlighting their potential in biomedical diagnostics. This work builds on these advancements, integrating the capabilities of NV centers with the detection of miRNAs, a critical biomarker in disease diagnosis.

This study uses nitrogen-vacancy (NV) centers in diamond to detect microRNA-21 (miR-21), a miRNA upregulated in several cancers. The approach is based on measuring the magnetic noise generated by Mn²⁺ ions, which are electrostatically attracted to the negatively charged phosphate backbone of miR-21 and the negatively charged surface of the diamond. The presence of miR-21 near the diamond surface increases the local concentration of Mn²⁺ ions, thereby enhancing the magnetic noise, which is detected by measuring the longitudinal spin-lattice relaxation time (T₁) of the NV centers. This magnetic noise measurement circumvents the limitations imposed by Debye screening of electric fields. **Experimental Setup:** A single-crystal diamond sample with NV centers implanted approximately 7 nm below the (100) surface was used. The diamond surface was oxygenated using a Piranha solution to create negatively charged functional groups, facilitating adsorption of miR-21 and Mn²⁺ ions. A microfluidic device allowed for precise control and sequential injection of different solutions. A 532 nm laser was used to initialize and readout the NV spin states, and a pulse sequence was employed to measure the T₁ relaxation time. Spin contrast, calculated from measurements at two different time points (10 µs and 400 µs), was used as a measure of the magnetic noise. The experiments involved sequential injection of solutions: (1) 1 mM EDTA (to neutralize the surface), (2) 5 mM MnCl₂ and 10 mM NaCl (to introduce Mn²⁺ ions), (3) 1 µM miR-21 in the MnCl₂/NaCl solution (to introduce miR-21), and (4) 1 mM EDTA (to remove Mn²⁺ ions). The change in spin contrast upon miR-21 injection was taken as the signal. **Molecular Dynamics (MD) Simulations:** All-atom MD simulations were performed to understand the interaction between miR-21, Mn²⁺ ions, and the diamond surface. The simulations used a system with two diamond slabs, miR-21, water molecules, and Mn²⁺, Na⁺, and Cl⁻ ions at concentrations matching the experimental conditions. The diamond surface was functionalized with hydroxyl, epoxy, carbonyl, ether, and carboxyl groups based on XPS measurements. Multiple simulations were run with varying surface charges and compositions. The simulations tracked the adsorption of miR-21 onto the diamond surface, the distribution of Mn²⁺ ions near the surface, and the magnetic field experienced by the NV centers. The magnetic noise (⟨B²⟩) was calculated from the simulated Mn²⁺ ion distribution. The simulation results were then compared to the experimental measurements of NV spin relaxation rates.

The experimental results demonstrated a clear increase in the NV spin relaxation contrast upon introduction of miR-21, indicating an increase in magnetic noise due to the accumulation of Mn²⁺ ions near the diamond surface. The magnitude of this increase correlated with the concentration of miR-21. A limit of detection (LOD) of 10 pM (120 attomoles) was achieved. Control experiments with no Mn²⁺ present showed no significant change in spin contrast upon miR-21 introduction, confirming that the signal was due to magnetic noise rather than electric noise. The MD simulations confirmed the experimental findings. They showed that the presence of miR-21 near the diamond surface led to a significant increase in the local concentration of Mn²⁺ ions. The simulations indicated that a single adsorbed miR-21 molecule accumulates approximately 8-9 Mn²⁺ ions within 4 nm of the surface. The calculated magnetic noise (⟨B²⟩) was consistent with the experimentally observed increase in NV spin relaxation rate. The simulations also revealed the importance of the diamond surface charge and composition on miR-21 adsorption, affecting the adsorption rate but not the stability of adsorption after it occurred.

The results demonstrate a novel quantum sensing method for label-free detection of miRNAs, overcoming the limitations of Debye screening. By measuring magnetic noise from counterions, instead of directly measuring the partially screened charge of the biomolecule, the method achieves high sensitivity and avoids the need for labeling or amplification. The sensitivity of 10 pM represents a significant advancement compared to existing label-free methods. The MD simulations provide a clear molecular-level understanding of the underlying mechanism, confirming that miR-21 adsorption leads to an increase in Mn²⁺ concentration near the diamond surface. The agreement between the experimental and simulation results strengthens the validity of the proposed mechanism. The findings have broad implications for various applications where the sensitive detection of polyelectrolytes is necessary. The method's potential extends beyond miRNA detection to other types of charged biomolecules and synthetic polyelectrolytes, opening up new possibilities in fields like water treatment, filtering, enhanced oil recovery, food science, cosmetics, batteries, and other biomedical applications.

This study successfully demonstrated a quantum sensing method for label-free detection of microRNA-21 using NV centers in diamond. The method is based on the measurement of magnetic noise generated by Mn²⁺ counterions that accumulate near the diamond surface due to interaction with miR-21. This approach circumvents the limitation imposed by Debye screening, achieving a limit of detection of 10 pM. Molecular dynamics simulations supported the experimental results and provided valuable insights into the underlying mechanisms. This method holds great promise for various applications requiring sensitive, label-free detection of polyelectrolytes, particularly in biomedical diagnostics. Future work could focus on enhancing sensitivity further by optimizing the NV center depth, employing more sophisticated pulse sequences for noise cancellation, and adapting the technique for high-throughput analysis, such as in microarray-based detection.

The current study focuses on a single miRNA, miR-21. Further research is needed to evaluate the generality of the method for other miRNAs and biomolecules. The MD simulations, while valuable, relied on several assumptions and approximations. Improving the accuracy and refinement of the simulations, particularly regarding the dynamics of Mn²⁺ ions bound to RNA, could enhance the quantitative agreement between simulations and experimental results. The experiments were performed under specific buffer conditions and pH values. Exploring the robustness of the method under a wider range of conditions is crucial for practical applications. The potential influence of silicon contamination in the diamond sample on the interaction of Mn²⁺ ions and miR-21 with the diamond surface warrants further investigation.