An optical quantum biological detection system and method based on a digital microfluidic platform

Abstract

This invention relates to the field of photonic quantum biological detection technology, and particularly to a photonic quantum biological detection system and method based on a digital microfluidic platform. The system includes a droplet loading module, a droplet manipulation module, a photomagnetic co-modulation module, a signal acquisition module, and a signal processing module. The droplet loading module loads an engineered bacterial suspension expressing a magnetically sensitive protein and a solution of a paramagnetic substance to be tested into a digital microfluidic chip, forming independent engineered bacterial droplets and analyte droplets. The droplet manipulation module applies a voltage sequence to the electrode array of the digital microfluidic chip via the digital microfluidic platform. This invention achieves high-precision programmable manipulation of droplets through a digital microfluidic platform, precisely mixing and positioning the engineered bacteria expressing the magnetically sensitive protein with the paramagnetic substance to be tested. This overcomes the limitations of solid-state probes, such as difficulty in cell internalization, high heterogeneity, and inability to integrate genes, achieving quantum sensing at the molecular level of living cells.

Description

Technical Field

[0001] This invention relates to the field of photonic quantum biological detection technology, and in particular to a photonic quantum biological detection system and method based on a digital microfluidic platform. Background Technology

[0002] Quantum sensing technology, with its extreme sensitivity to environmental perturbations in quantum coherent states, has become a revolutionary tool for probing microscopic thermodynamic and electromagnetic processes. In the field of biosensing, fluorescent nanodiamonds (FNDs) based on nitrogen-vacancy centers in diamond have long been considered the "gold standard" for nanoscale cell thermometry and magnetic detection. However, as exogenous inorganic solid-state probes, FNDs face limitations such as difficulties in efficient cell internalization, measurement errors caused by particle heterogeneity, and the inability to participate in gene regulatory networks, restricting their in-situ in vivo applications. To overcome the interface barrier between solid-state sensors and biological systems, gene-encoded magnetic proteins have emerged. Among them, the MagLOV protein, developed through directed evolution, is inspired by the cryptochrome radical pair mechanism in migratory bird magnetic navigation. It utilizes the photochemical cycle of flavin cofactors to generate spin-related radical pairs, whose quantum coherent evolution response to weak magnetic fields is converted into fluorescent signals. This gene-programmable characteristic allows it to be directly expressed in chassis such as E. coli, achieving molecular-level fusion and effectively solving the biocompatibility and integration problems of traditional solid-state probes.

[0003] However, achieving high-precision quantum sensing in the complex environment of living cells still faces technological bottlenecks. The molecular crowding environment within biological systems, the inherent autofluorescence background, and optical scattering often result in extremely low signal-to-noise ratios for magnetic field effect signals. Furthermore, the unavoidable photobleaching effect of fluorescent proteins under continuous light excitation, and the heterogeneity between individual cells, severely restrict long-term dynamic observation and quantitative analysis. Traditional static microscopy struggles to achieve precise manipulation of the microenvironment of individual cells, while existing continuous flow microfluidic systems, although capable of averaging differences through flow, lack the flexibility for programmable chemical perturbation of individual samples and long-term fixed-point monitoring. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a photonic quantum biological detection system based on a digital microfluidic platform, comprising a droplet loading module, a droplet manipulation module, an optomagnetic co-modulation module, a signal acquisition module, and a signal processing module. The droplet loading module loads an engineered bacterial suspension expressing a magnetically sensitive protein and a solution of a paramagnetic substance to be tested into a digital microfluidic chip, forming independent engineered bacterial droplets and analyte droplets. The droplet manipulation module applies a voltage sequence to the electrode array of the digital microfluidic chip via the digital microfluidic platform, driving the analyte droplets and engineered bacterial droplets to merge and mix using the dielectric wetting principle, and positioning the mixed detection droplets in the detection area. The optomagnetic co-modulation module applies fluorescent excitation light and a periodically modulated magnetic field to the detection area. The signal acquisition module continuously acquires the dynamic fluorescence signal generated by the magnetically sensitive protein in the detection droplets in response to the periodic magnetic field modulation. The signal processing module performs lock-in amplification on the acquired dynamic fluorescence signal, extracts the signal amplitude with the same frequency as the magnetic field modulation, and determines the concentration of the paramagnetic substance to be tested based on the correspondence between the signal amplitude and a preset standard curve.

[0005] Furthermore, the optical-magnetic co-modulation module includes a fluorescence excitation block, an objective lens, a digital microfluidic chip, an inverted fluorescence microscope, an sCMOS camera, and a light-emitting diode (LED) light source. The inverted fluorescence microscope serves as the optical imaging path and works in conjunction with the LED light source to complete the excitation-collection process. The LED provides stable excitation light of a specific wavelength. The excitation light passes through the fluorescence excitation block and is focused onto the droplet reaction region formed on the digital microfluidic chip by an objective lens with a certain magnification. The inverted fluorescence microscope collects the fluorescence signal generated in the droplet reaction region. The sCMOS camera periodically captures the fluorescence signal at the location of the droplet reaction region.

[0006] Furthermore, the digital microfluidic chip is used as a reaction chamber; the digital microfluidic chip includes an electrode substrate and a cover plate; the electrode substrate is provided with a plurality of driving electrode arrays, at least one reagent storage area and a droplet manipulation channel; the geometric dimensions of the digital microfluidic chip are in the range of electrode size of 250μm×250μm, droplet manipulation channel height of 50-150μm and dielectric layer thickness of 300nm.

[0007] Furthermore, the droplet manipulation module includes an electronic drive unit, a flexible printed circuit board, and a software interface; engineered E. coli bacteria expressing the magnetic fluorescent protein MagLOV are combined with paramagnetic metal ions gadolinium (Gd). 3+The droplets are controlled by an electronic drive unit via a software interface to apply a voltage sequence to the electrode array of the digital microfluidic chip through a flexible printed circuit board. The principle of dielectric wetting is used to drive, transport, mix, position and refresh the droplets on the digital microfluidic chip. The droplets are manipulated from the loading area to the detection area through the electrode array, and automatically removed and replenished with fresh droplets after detection.

[0008] Furthermore, the optical-magnetic co-modulation module also includes a controllable magnetic field generating unit and a magnetic field strength adjustment unit; the controllable magnetic field generating unit is composed of an iron-core electromagnet; the magnetic field strength adjustment unit includes a signal generator, a power amplifier, and a power adapter; the signal generator outputs a pulse width modulation (PWM) signal with adjustable frequency and duty cycle, which is amplified by the power amplifier and then supplied with stable power by the power adapter to drive the iron-core electromagnet to generate a periodic modulated magnetic field.

[0009] Furthermore, the engineered Escherichia coli containing the MagLOV magnetic protein-expressing bacterial suspension was constructed using the pET-28a(+) vector, and the MagLOV gene was inserted into the NcoI / XhoI site. After sequencing verification, it was transformed into Escherichia coli BL21(DE3) strain.

[0010] Furthermore, the paramagnetic metal ions include at least one of free gadolinium ions and complexed gadolinium ions; the concentration range of the paramagnetic metal ion solution is 0.01-100 mM.

[0011] This invention also provides a photonic quantum biological detection method based on a digital microfluidic platform, comprising:

[0012] Step S1: The engineered bacterial suspension containing MagLOV magnetic sensitive protein and the solution of the paramagnetic substance to be tested are loaded into the digital microfluidic chip to form independent engineered bacterial droplets and test substance droplets.

[0013] Step S2: Apply a voltage sequence to the electrode array of the digital microfluidic chip through the digital microfluidic platform, drive the test analyte droplets and engineered bacterial droplets to merge and mix using the dielectric wetting principle, and position the mixed detection droplets to the detection area;

[0014] Step S3: Apply fluorescence excitation light and periodically modulated magnetic field to the detection area, and continuously acquire dynamic fluorescence signals generated by the magnetic protein in the detection drop in response to the periodic magnetic field modulation.

[0015] Step S4: The acquired dynamic fluorescence signal is amplified by lock-in processing, and the signal amplitude with the same frequency as the magnetic field modulation frequency is extracted. Based on the correspondence between the signal amplitude and the preset standard curve, the concentration of the paramagnetic substance to be tested is determined.

[0016] Furthermore, the periodically modulated magnetic field in step S2 is generated in the following way:

[0017] A pulse width modulation signal is output from a signal generator, amplified by a power amplifier, and then drives an electromagnet to generate the periodic modulated magnetic field.

[0018] Furthermore, the specific method for continuously acquiring dynamic fluorescence signals in step S3 is as follows:

[0019] Incident light is emitted by a light-emitting diode (LED) source, passes through a fluorescence excitation block, and is focused onto the detection droplet region by an objective lens. An image sensor periodically captures fluorescence signals at the location of the droplet reaction zone.

[0020] The beneficial effects of the present invention are as follows: (1) The present invention achieves high-precision programmable control of droplets through a digital microfluidic platform, accurately mixes and positions engineered bacteria expressing magnetic sensitive proteins with the paramagnetic substances to be tested, overcomes the limitations of solid probes in cell internalization difficulties, large heterogeneity and inability to integrate genes, and realizes quantum sensing of molecular-level fusion of living cells. Using MagLOV-expressing engineered bacteria as quantum sensing units, combined with digital microfluidic chips, the present invention achieves high-precision control of droplets, overcomes the limitations of solid probes in cell internalization low heterogeneity and inability to integrate genes, and realizes molecular-level fusion of living cells. (2) The present invention uses lock-in amplification to extract high signal-to-noise ratio magnetic field effect signals, and realizes label-free and non-destructive quantitative detection of paramagnetic ions and clinical MRI contrast agents. It can effectively filter out background noise, which is beneficial to improve the measurement sensitivity in noisy biological environments. Secondly, by utilizing the programmability of digital microfluidics, a measurement method based on droplet renewal is constructed: the photobleached droplets are automatically removed from the detection area and droplets containing fresh bacteria are replenished in real time. It maintains extremely high measurement stability on a macroscopic time scale, overcomes the limitation of short lifespan of traditional biosensors, and eliminates the influence of thermal drift in long-term measurements. (3) The present invention automatically removes the photobleached droplets and replenishes fresh droplets through a droplet dynamic refresh mechanism, ensuring signal stability for long-term measurements, overcoming the limitation of short lifespan of traditional biosensors, and providing a standardized and automated detection platform for the accurate characterization of microscopic spin dynamics in complex biological environments. Attached Figure Description

[0021] Figure 1 This is a basic flowchart of a photonic biological detection system based on a digital microfluidic platform in Example 1.

[0022] Figure 2 This is a schematic diagram of the digital microfluidic chip used to perform the droplet manipulation step in Example 1.

[0023] Figure 3 For example 2, different concentrations of paramagnetic ions (Gd) were considered. 3+The time-domain waveform of a typical modulated fluorescence signal is recorded.

[0024] Figure 4 For based on Figure 2 The average phase-locked amplitude calculated from the data varies with Gd 3+ Quantitative response curve of concentration change.

[0025] Figure 5 For example 2, different paramagnetic ions (Ni) 2+ Typical modulated fluorescence signal time-domain waveform of concentration recording.

[0026] Figure 6 For based on Figure 4 The average phase-locked amplitude calculated from the data varies with Ni 2+ Quantitative response curve of concentration change.

[0027] Figure 7 This is a typical modulated fluorescence signal time-domain waveform diagram for detecting the clinical macrocyclic contrast agent DOTA-Gd in Example 3.

[0028] Figure 8 This is the concentration-response curve for detecting the clinical macrocyclic contrast agent DOTA-Gd in Example 3.

[0029] Figure 9 This is a typical modulated fluorescence signal time-domain waveform diagram for detecting the clinical macrocyclic contrast agent Gadobutrol in Example 4.

[0030] Figure 10 This is the concentration-response curve for testing the clinical macrocyclic contrast agent Gadobutrol in Example 4. Detailed Implementation

[0031] Example 1, an embodiment of the present invention, provides a photonic quantum biological detection system based on a digital microfluidic platform, including a droplet loading module, a droplet manipulation module, an optical-magnetic co-modulation module, a signal acquisition module, and a signal processing module;

[0032] The droplet loading module is used to load the engineered bacterial suspension expressing the magnetically sensitive protein and the solution of the paramagnetic substance to be tested into the digital microfluidic chip, forming independent engineered bacterial droplets and analyte droplets. The droplet loading module corresponds to the reservoir area and sample application interface of the digital microfluidic chip, introducing reagents into the chip cavity via a pipette or autosampler. The droplet loading module is the physical interface and structural unit used to transform macroscopically sized reagent samples into controllable discrete microdroplets within the digital microfluidic chip, enabling the input of raw materials for the detection reaction. As the system's input, the droplet loading module receives the externally prepared engineered bacterial suspension and the solution of the paramagnetic substance to be tested, guides them to the reservoir area of ​​the digital microfluidic chip, and provides the initial operating objects for the subsequent droplet manipulation module.

[0033] The droplet loading module specifically includes a chip reservoir, a sample dispensing port, and a sealing cover. The chip reservoir is a recessed structure located at the edge of the digital microfluidic chip, used to temporarily store the loaded reagents; the sample dispensing port is a through-hole that penetrates the cover, used for inserting a pipette tip to inject reagents; the sealing cover is a transparent structure covering the reservoir, used to prevent reagent evaporation and external contamination.

[0034] Operators use pipettes to aspirate the suspension of engineered bacteria expressing MagLOV magnetic protein and the solution of the paramagnetic substance to be tested, injecting them into the chip's reservoir through the sample wells. Under dielectric wetting or capillary action, the reagents fill to the edge of the electrode array, awaiting subsequent droplet cutting and actuation operations.

[0035] Combination Figure 2 A digital microfluidic chip is used as the reaction chamber. The digital microfluidic chip includes a top substrate and a bottom substrate. The top substrate is made of a transparent material with indium tin oxide (ITO) conductive glass in the middle. A hydrophobic layer is disposed inside the bottom substrate, and an electrode array can be placed in the hydrophobic layer. The hydrophobic layer prevents droplets from directly contacting the electrodes, thus preventing droplet movement and achieving dielectric wetting. The electrode array is disposed on the electrode substrate, which has at least one reagent reservoir and a droplet manipulation channel. The geometric dimensions of the digital microfluidic chip range from 250 μm × 250 μm for the electrodes, 50-150 μm for the droplet manipulation channel height, and 300 nm for the dielectric layer thickness.

[0036] The droplet manipulation module applies a voltage sequence to the electrode array of the digital microfluidic chip via a digital microfluidic platform. Utilizing the dielectric wetting principle, it drives the analyte droplets and engineered bacterial droplets to merge and mix, and then positions the mixed detection droplets to the detection area. The droplet manipulation module corresponds to the chip driving system and includes an electronic driving unit, a flexible printed circuit board, and control software. The droplet manipulation module is the core actuator for generating, transporting, merging, mixing, and positioning droplets based on the dielectric wetting principle (EWOD), enabling automated microfluidic operation of the detection process.

[0037] In this embodiment, the droplet manipulation module receives a sequence of instructions from the signal processing module (control software), applies a driving voltage to a specific electrode of the digital microfluidic chip, changes the droplet contact angle, and thus drives its movement. This achieves precise mixing and detection of the engineered bacterial droplet and the analyte droplet, and works in conjunction with the optomagnetic co-modulation module to ensure the droplet is in the optimal observation position. Engineered E. coli containing the magnetofluorescent protein MagLOV is mixed with the paramagnetic metal ion gadolinium (Gd). 3+ The software interface controls the electronic drive unit to apply a voltage sequence to the electrode array of the digital microfluidic chip via a flexible printed circuit board. Utilizing the dielectric wetting principle, the droplets are driven, transported, mixed, positioned, and refreshed on the digital microfluidic chip. The droplets are manipulated from the loading area to the detection area via the electrode array, and automatically removed and replenished with fresh droplets after detection. The paramagnetic metal ion gadolinium includes at least one of free gadolinium ions and complexed gadolinium ions; the concentration range of the paramagnetic metal ion gadolinium solution is 0.01-100 mM.

[0038] The droplet manipulation module specifically includes an electronic drive unit, a flexible printed circuit board (FPC), a digital microfluidic chip electrode array, and a control software unit. The electronic drive unit is a high-voltage switching array circuit used to convert low-voltage control signals into high-voltage AC signals (typically 100V-300V) capable of driving the droplets. The flexible printed circuit board is a flexible conductive line connecting the electronic drive unit and the chip electrodes, used to ensure reliable transmission of electrical signals. The digital microfluidic chip electrode array is a two-dimensional planar structure composed of multiple independently controlled square electrodes, used to manipulate the droplet path through changes in the electric field distribution. The control software unit is a human-machine interface program running on a host computer, used to edit the droplet manipulation path, set the mixing time, and the refresh frequency.

[0039] The control software unit controls the output voltage sequence of the electronic drive unit according to a preset program. First, it activates the electrodes adjacent to the liquid storage area, pulling out the droplet and cutting it into independent units. Then, by activating the electrodes along the path in sequence, it drives the engineered bacteria droplet and the test substance droplet to move to the mixing point respectively. Finally, it controls the two droplets to merge and accelerates the diffusion and mixing through oscillation, positioning the mixed droplet in the detection area directly below the microscope objective.

[0040] The opto-magnetic co-modulation module is used to apply fluorescent excitation light and a periodically modulated magnetic field to the detection area. The module includes an optical excitation unit and a magnetic field generation unit. The optical excitation unit includes an LED light source, a fluorescent excitation block, and an objective lens. The magnetic field generation unit includes a signal generator, a power amplifier, and an iron-core electromagnet. The LED light source emits 460nm blue light; the fluorescent excitation block filters stray light and selects a specific excitation wavelength; the objective lens focuses the excitation light onto the chip's detection area. The signal generator generates a pulse-width modulation (PWM) signal with a specific frequency (e.g., 1Hz-10Hz) and duty cycle; the power amplifier amplifies the PWM signal to drive the electromagnet; the iron-core electromagnet generates a periodically switching magnetic field, with its magnetic pole head positioned close to the digital microfluidic chip's detection area.

[0041] The optical-magnetic co-modulation module also includes an inverted fluorescence microscope, an sCMOS camera, and a light-emitting diode (LED) light source. The inverted fluorescence microscope acts as the optical imaging path, working in conjunction with the LED light source to complete the excitation-collection process. The LED provides stable excitation light at a specific wavelength. The excitation light passes through a fluorescence excitation block and is focused onto the droplet reaction region formed on the digital microfluidic chip by an objective lens with a certain magnification. The inverted fluorescence microscope collects the fluorescence signal generated in the droplet reaction region. The sCMOS camera periodically captures the fluorescence signal at the location of the droplet reaction region. The inverted fluorescence microscope is a Nikon Eclipse Ti2-U.

[0042] The optomagnetic co-modulation module also includes a controllable magnetic field generation unit and a magnetic field strength adjustment unit. The controllable magnetic field generation unit consists of an iron-core electromagnet; the magnetic field strength adjustment unit includes a signal generator, a power amplifier, and a power adapter. The signal generator outputs a pulse-width modulation (PWM) signal with adjustable frequency and duty cycle. After being amplified by the power amplifier, the signal is stably powered by the power adapter, driving the iron-core electromagnet to generate a periodically modulated magnetic field. The optomagnetic co-modulation module is a hardware combination that provides a physical environment of excitation light of a specific wavelength and a periodically changing magnetic field to the detection area, used to induce the MagLOV magnetic protein to generate a detectable dynamic fluorescence signal.

[0043] The optomagnetic co-modulation module is activated after the droplet is positioned. The optical part excites the droplet to produce fluorescence, while the magnetic part applies a periodic magnetic field to modulate the state of free radical pairs. The synergistic effect of these two components causes the fluorescence intensity to fluctuate regularly in sync with the magnetic field, providing a high-quality time-varying signal source for the signal acquisition module. Once the mixed droplet is positioned stably, the LED light source is turned on, and blue light is focused onto the droplet through the objective lens. Simultaneously, the signal generator outputs a square wave signal, which drives an electromagnet via a power amplifier to generate a periodic magnetic field, such as "on for 1 second - off for 1 second". During the alternation of the magnetic field being turned on and off, the fluorescence intensity of the MagLOV protein within the droplet changes synchronously.

[0044] The signal acquisition module is used to continuously acquire the dynamic fluorescence signal generated by the response of the magnetosensitive protein in the droplet to periodic magnetic field modulation. The signal acquisition module is a collection of photoelectric conversion devices used to capture optical images of the detection area and convert them into digital signals for transmission to the processor, enabling high-sensitivity recording of dynamic fluorescence signals. The signal acquisition module receives the fluorescence signal excited by the photomagnetic co-modulation module, converts the light signal into an image sequence through the optical imaging system, and transmits it in real time to the signal processing module for subsequent analysis. Its acquisition frequency must match the magnetic field modulation frequency to satisfy the Nyquist sampling theorem.

[0045] The signal acquisition module specifically includes an imaging objective, a fluorescence filter, an sCMOS camera, and an image acquisition card. The imaging objective is used to collect the fluorescence signal emitted by the droplet; the fluorescence filter is used to filter out the excitation light background, allowing only the fluorescence in the emission band to pass through; the sCMOS camera is a scientific-grade complementary metal-oxide-semiconductor camera used to capture fluorescence images at high frame rate and low noise; and the image acquisition card is used to achieve high-speed data transmission between the camera and the computer.

[0046] In practice, the sCMOS camera was set to an exposure time of 100 ms and an interval time of 100 ms, continuously capturing a 120-second image sequence. The image sequence was saved to computer memory in real time, recording the complete process of fluorescence intensity changes over time and with the magnetic field state. The signal processing module was used to perform lock-in amplification on the acquired dynamic fluorescence signal, extracting the signal amplitude at the same frequency as the magnetic field modulation. Based on the correspondence between the signal amplitude and a preset standard curve, the concentration of the paramagnetic substance to be tested was determined.

[0047] The engineered Escherichia coli strain containing the MagLOV magnetic protein expressed in the suspension was constructed using the pET-28a(+) vector. The MagLOV gene was inserted into the NcoI / XhoI site, and after sequencing verification, it was transformed into Escherichia coli strain BL21(DE3).

[0048] The signal processing module is a set of computational units and algorithms that process and analyze the acquired raw image data and output the final detection results, enabling signal extraction and quantitative analysis. The signal processing module receives the image sequence transmitted by the signal acquisition module, first calculating the average gray value of the region of interest (ROI) in each frame to generate a fluorescence intensity time-series curve, then using a lock-in amplification algorithm to extract the magnetic field effect signal, and finally calculating the concentration of the analyte according to a preset model.

[0049] The signal processing module specifically includes an image processing unit, a lock-in amplification algorithm unit, and a quantitative analysis unit. The image processing unit is used for image background subtraction, ROI selection, and grayscale value calculation; the lock-in amplification algorithm unit is used to perform cross-correlation calculations between the fluorescence intensity time-series signal and the magnetic field reference signal to extract the amplitude and phase of the same-frequency components; the quantitative analysis unit is used to store standard curve data and map the calculated signal amplitude to the concentration value of the paramagnetic substance to be measured.

[0050] The processor executes a computer program to perform the following steps: read the image sequence acquired by the sCMOS camera; select the droplet center region as the ROI and calculate the average fluorescence intensity I(t); acquire the magnetic field modulation reference signal R(t) (square wave); calculate the cross-correlation function to obtain the phase-locked amplitude; query the pre-stored concentration-amplitude standard curve and calculate the Gd to be measured through interpolation. 3+ The concentration is calculated and the results are displayed on the software interface.

[0051] In this embodiment, MagLOV-expressing engineered bacteria are used as quantum sensing units, combined with a DMF chip to achieve high-precision droplet manipulation. This overcomes the limitations of solid-state probes, such as low cell internalization, high heterogeneity, and inability to integrate genes, enabling molecular-level fusion of live cells. Periodic magnetic field modulation and lock-in amplification are used to extract high signal-to-noise ratio magnetic field effect signals, achieving label-free and non-destructive quantitative detection of paramagnetic ions and clinical MRI contrast agents. The DMF droplet dynamic refresh mechanism ensures long-term signal stability, providing a standardized and automated platform and enhancing the accurate characterization of microscopic spin dynamics in complex biological environments.

[0052] Example 2 is another embodiment of the present invention. This embodiment is based on the previous embodiment, but differs from the previous embodiment in that it provides a photonic quantum biological detection method based on a digital microfluidic platform, comprising:

[0053] Step S1: The engineered bacterial suspension containing MagLOV magnetic sensitive protein and the solution of the paramagnetic substance to be tested are loaded into the digital microfluidic chip to form independent engineered bacterial droplets and analyte droplets. In this step, the engineered bacterial suspension containing the magnetic sensitive protein is a liquid suspension system containing Escherichia coli engineered bacteria that can respond to changes in magnetic field and generate fluorescent signals, which is used as a biological quantum sensing unit.

[0054] The engineered bacterial suspension, prepared by the bacterial suspension preparation module, is loaded into the reservoir of the digital microfluidic chip. After droplet generation, individual droplets are formed, awaiting subsequent mixing and detection. The engineered bacterial suspension specifically includes engineered *E. coli* bacteria expressing the MagLOV magnetic protein, HEPES buffer, and necessary culture medium components. The MagLOV magnetic protein is a fusion protein constructed using genetic engineering technology. It contains a flavin adenine dinucleotide (FAD) binding domain and can generate free radical pairs under blue light excitation, thus responding to changes in magnetic fields. The HEPES buffer is used to maintain a stable pH (pH 7.4) in the droplet environment to ensure bacterial activity. The paramagnetic analyte solution is a liquid system containing the paramagnetic ions to be detected, used to mix with the engineered bacterial suspension for quantitative analysis.

[0055] The solution of the paramagnetic substance to be tested is loaded into another reservoir area of ​​the chip, forming independent droplets, which are then merged with the engineered bacterial droplets under the control of a digital microfluidic system. The solution of the paramagnetic substance to be tested specifically includes paramagnetic metal ions and a solvent. In this embodiment, the paramagnetic metal ions are gadolinium ions (Gd). 3+ The solvent is HEPES buffer. The test solutions are prepared with concentration gradients according to experimental requirements, for example, 2 μM, 20 μM, 200 μM, 2 mM, 20 mM, and 200 mM.

[0056] Prepare a suitable 10mM HEPES buffer (pH 7.4), and use this buffer to prepare an engineered E. coli suspension expressing MagLOV, adjusting the OD600 to 1. Simultaneously, prepare suspensions containing different concentrations of Gd... 3+ The engineered bacterial suspension and the test solution were loaded into the reservoir area of ​​the digital microfluidic chip cavity through dedicated inlets, forming multiple independent bacterial droplets. These droplets were then incubated on the digital microfluidic chip for 10–20 min to allow the bacteria to stably locate in the detection area.

[0057] Step S2 involves applying a voltage sequence to the electrode array of the digital microfluidic chip via a digital microfluidic platform. This utilizes the dielectric wetting principle to drive the analyte droplets and engineered bacterial droplets to merge and mix, and then positioning the mixed detection droplets within the detection area. The periodically modulated magnetic field is generated by a pulse-width modulation signal output from a signal generator, which is amplified by a power amplifier to drive an electromagnet to generate the periodically modulated magnetic field.

[0058] Digital microfluidic chips are miniaturized chip devices that manipulate discrete droplets based on the principle of dielectric wetting, providing a microenvironment for droplet reactions. As a reaction vessel, the digital microfluidic chip receives the loaded reagents and, driven by an electrode array, realizes the movement, merging, and positioning of droplets. The principle of dielectric wetting is a physical mechanism that drives the movement of conductive droplets by changing the electrowetting tension of the electrode surface, enabling precise droplet manipulation. In this embodiment, when a voltage is applied to a specific electrode, the wettability of the electrode surface changes, resulting in a difference in contact angle between the two ends of the droplet, thereby driving the droplet to move towards the electrode to which the voltage is applied.

[0059] The droplet manipulation process specifically includes droplet generation, transport, merging, mixing, and positioning. First, by controlling the on / off state of the electrode array, upgraded engineered bacterial droplets and analyte droplets are separated from the reservoir. Then, the two droplets are driven along a preset path to a designated mixing point. At the mixing point, the two droplets contact and merge. By controlling the voltage switching of adjacent electrodes, the merged droplets are made to reciprocate or oscillate between the electrodes to accelerate homogenization. Finally, the mixed detection droplet is transported to the detection area of ​​the optical observation platform (e.g., directly below the microscope objective) and kept stationary.

[0060] Step S2 also includes a droplet refresh step, which involves removing photobleached droplets from the detection area and replenishing them with fresh droplets after or during detection to maintain signal stability over long periods. In this embodiment, the droplet refresh step is executed by an electronically driven unit controlled by a software interface. When the fluorescence signal decays to a preset threshold or a measurement cycle is completed, the system automatically drives the detection droplets out of the detection area to the waste liquid area and then drives new engineered bacterial droplets from the storage area to mix and position with the analyte droplets. The electrode array is controlled by software, utilizing the dielectric wetting principle to drive droplets containing different concentrations of Gd... 3+ Ionic droplets and engineered bacterial droplets are merged at a designated mixing point to achieve precise mixing within the chip. After mixing, the droplets are automatically transported to the detection area and kept stationary. If long-term monitoring is required, after detection, the photobleached droplets are automatically removed from the detection area and replenished with droplets containing fresh bacteria to maintain measurement stability.

[0061] Step S3 involves applying fluorescent excitation light and a periodically modulated magnetic field to the detection area, while continuously acquiring the dynamic fluorescence signal generated by the magnetically sensitive protein in the detection droplet in response to the periodic magnetic field modulation. The specific method for continuously acquiring the dynamic fluorescence signal is as follows: the incident light is emitted by a light-emitting diode (LED) source, passes through a fluorescence excitation block, and is focused onto the detection droplet area by an objective lens. An image sensor periodically captures the fluorescence signal at the droplet reaction area. The fluorescent excitation light is a specific wavelength of light capable of exciting the MagLOV magnetically sensitive protein to produce fluorescence, used to induce the sensing unit to generate a signal. The fluorescent excitation light is emitted by the light source of the optical observation platform, passes through the optical path system, and is focused onto the detection droplet area, exciting the MagLOV protein in the engineered bacteria to produce fluorescence. Specifically, the fluorescent excitation light is a blue LED source with a wavelength of 460 nm. The incident light passes through the fluorescence excitation block (filter) and is focused onto the detection droplet area formed on the digital microfluidic chip by an objective lens.

[0062] Dynamic fluorescence signals are a sequence of fluorescence intensity changes over time, used to reflect the effect of magnetic field modulation on fluorescence. Dynamic fluorescence signals are continuously acquired by an image sensor on an optical observation platform, recording the difference in fluorescence intensity of the droplet when the magnetic field is on and off. The specific method for continuously acquiring dynamic fluorescence signals is as follows: incident light is emitted from a light source, passes through a fluorescence excitation block, and is focused onto the detection droplet region by an objective lens. An sCMOS camera then periodically captures fluorescence signals at specific locations. The imaging parameters are set as follows: exposure time 100 ms, interval time 100 ms, and a total acquisition period of 120 s. The stable region at the center of the droplet is selected as the imaging area, and the change in fluorescence intensity with time and magnetic field modulation is recorded. Under 460 nm blue light excitation, an inverted fluorescence microscope combined with an sCMOS camera periodically captures the droplet fluorescence signal. Simultaneously, the magnetic field modulation component is activated, applying a periodically modulated magnetic field.

[0063] A periodically modulated magnetic field is a magnetic field whose intensity varies periodically with time. It is used to modulate the state of free radical pairs in the MagLOV protein, thereby producing detectable changes in fluorescence intensity. The periodically modulated magnetic field is generated by a magnetic field control component, and its frequency and duty cycle can be adjusted according to experimental requirements. The presence or absence of the magnetic field alters the intersystem crossing rate of MagLOV protein free radical pairs, thus affecting the fluorescence quantum yield and producing fluorescence intensity fluctuations synchronized with the magnetic field. Specifically, the periodically modulated magnetic field is generated by a pulse-width modulation (PWM) signal output from a signal generator, the frequency and duty cycle of which are adjustable. After being amplified by a power amplifier, the signal drives an iron-core electromagnet to generate a periodically switching magnetic field. The magnetic field strength can be controlled by adjusting the gain of the power amplifier or the power supply voltage.

[0064] Step S4 involves performing lock-in amplification on the acquired dynamic fluorescence signal to extract the signal amplitude with the same frequency as the magnetic field modulation. Based on the correspondence between the signal amplitude and a preset standard curve, the concentration of the paramagnetic substance to be tested is determined. Lock-in amplification is a technique for extracting weak signals of specific frequencies from noise to improve the signal-to-noise ratio of detection. It utilizes the frequency of the magnetic field modulation as a reference signal to extract the effective signal component with the same frequency as the magnetic field from the acquired fluorescence signal, which contains a large amount of background noise.

[0065] The specific process of lock-in amplification includes: multiplying the acquired raw fluorescence intensity time-series signal with the reference signal (magnetic field modulation signal) and performing low-pass filtering. Since background noise is usually randomly distributed or frequency mismatched, it is filtered out, while the signal with the same frequency as the magnetic field modulation is retained and amplified.

[0066] Signal amplitude is the magnitude of the fluorescence signal that changes synchronously with magnetic field modulation, used to characterize the strength of the magnetic field effect. There is a correlation between signal amplitude and the concentration of the paramagnetic substance being measured. Paramagnetic substances (such as Gd) 3+ This will accelerate the relaxation process of MagLOV protein free radical pairs, resulting in a weakening of the magnetic field effect, i.e., a reduction in signal amplitude.

[0067] A preset standard curve is a working curve plotted by measuring the signal amplitude of a standard sample at a known concentration. It is used to convert the signal amplitude of an unknown sample into a concentration value. The preset standard curve is obtained by measuring a series of Gd samples at known concentrations. 3+ The signal amplitude of the standard solution was plotted. The curve typically exhibits a non-linear decay trend, and a mathematical model can be obtained through non-linear fitting.

[0068] like Figure 3 and Figure 4 As shown, Gd is increased successively 3+ Concentration was assessed, and the average phase-locked amplitude at each concentration was extracted, recording its trend with concentration. The acquired raw fluorescence signal was amplified using phase-locked processing, and the average amplitude value with the same frequency as the magnetic field modulation was extracted. The average phase-locked amplitude values ​​corresponding to each concentration were plotted point-by-point to form a concentration-response curve. This curve was then nonlinearly fitted to obtain a smooth decay trend curve. Based on this curve, quantitative analysis of samples with unknown concentrations can be achieved.

[0069] The processing module automatically acquires fluorescence image sequences from the sCMOS camera, calculates the average fluorescence intensity of the droplet region in each frame, and generates a time-series signal. Combined with a magnetically modulated time-series reference signal, a software algorithm is used to achieve phase-locked amplification and extract the average phase-locked amplitude. This replaces traditional manual recording and calculation methods, eliminating human error and improving the efficiency and accuracy of data processing.

[0070] This embodiment utilizes a digital microfluidic platform to achieve high-precision programmable manipulation of droplets, precisely mixing and positioning engineered bacteria expressing magnetosensitive proteins with the target paramagnetic substance. This overcomes the limitations of solid-state probes, such as difficulty in cell internalization, high heterogeneity, and inability to integrate genes, enabling quantum sensing with molecular-level fusion in living cells. Periodic magnetic field modulation and lock-in amplification techniques effectively filter out background noise in complex biological environments, improving the signal-to-noise ratio of magnetic field effect signals and achieving label-free, non-destructive quantitative detection of paramagnetic ions and clinical MRI contrast agents. A dynamic droplet refresh mechanism automatically removes photobleached droplets and replenishes them with fresh droplets, ensuring signal stability over long-term measurements. This overcomes the short lifespan limitations of traditional biosensors and provides a standardized and automated detection platform for the precise characterization of microscopic spin dynamics in complex biological environments.

[0071] Example 3, referring to Figure 3 and Figure 4 This is another embodiment of the present invention, based on the previous embodiment. In this embodiment, the signal acquisition step is performed. The optical observation platform includes an inverted fluorescence microscope, an sCMOS camera, and an LED light source. The inverted fluorescence microscope uses a Nikon Eclipse Ti2-U, and the light source is a 460 nm blue LED. The incident light passes through a 460 nm wavelength blue fluorescence excitation block, and is focused onto the droplet reaction region formed on the digital microfluidic chip by a 20X magnification objective lens. The sCMOS camera periodically captures the fluorescence signal of a specific detection area in the microfluidic droplet, generally selected in the central region after the droplet has stabilized, to capture the fluorescence intensity changes under periodic magnetic field modulation in real time.

[0072] The biodetection system materials in this embodiment include engineered *E. coli* strains expressing the MagLOV magnetic sensitive protein, paramagnetic metal ion solutions, and their concentration gradients. The engineered bacteria were constructed using the pET-28a(+) vector, with the MagLOV gene inserted into the NcoI / XhoI site, and transformed into *E. coli* strain BL21(DE3). The paramagnetic metal ion solutions were prepared with concentration gradients (1 μM–100 mM) according to experimental requirements. This embodiment provides a photonic quantum biodetection method based on a digital microfluidic platform for measuring the paramagnetic ion Gd. 3+ Concentration-dependent inhibition of the MagLOV magnetic protein fluorescence magnetic field effect (MFE) was achieved, and its paramagnetic relaxation enhancement (PRE) effect was quantitatively analyzed.

[0073] Example 3, the only difference between Example 3 and Example 2 is that the paramagnetic ion is replaced with Ni. 2+ Everything else is the same as in Example 1, and the time-domain waveform of the fluorescence signal and the concentration-response curve are as follows: Figure 5 and Figure 6 As shown.

[0074] Example 4, the only difference between Example 4 and Example 2 is that the paramagnetic ions are replaced with the clinical macrocyclic contrast agents DOTA-Gd and Gadobutrol. The fluorescence signal time-domain waveform and concentration-response curve are as follows. Figures 7 to 10 As shown.

[0075] according to Figures 5 to 10 This demonstrates that the systems and methods provided in Examples 1 and 2 can extract weak magnetic field effect signals even in complex biological droplet environments using phase-locked loop technology. By establishing a correlation between concentration and amplitude, accurate quantitative analysis can be achieved regardless of whether the paramagnetic substance is a metal ion or a clinical contrast agent. The droplet refresh mechanism of digital microfluidics ensures signal stability during long-term measurements.

Claims

1. A photonic quantum biological detection system based on a digital microfluidic platform, characterized in that, It includes a droplet loading module, a droplet manipulation module, an optical-magnetic co-modulation module, a signal acquisition module, and a signal processing module; The droplet loading module is used to load the engineered bacterial suspension expressing magnetic sensitive protein and the solution of the paramagnetic substance to be tested into the digital microfluidic chip respectively, forming independent engineered bacterial droplets and test substance droplets; The droplet manipulation module is used to apply a voltage sequence to the electrode array of the digital microfluidic chip through the digital microfluidic platform, drive the test droplet and engineered bacterial droplet to merge and mix using the dielectric wetting principle, and position the mixed detection droplet to the detection area; The optical-magnetic co-modulation module is used to apply fluorescent excitation light and a periodically modulated magnetic field to the detection area; The signal acquisition module is used to continuously acquire and detect the dynamic fluorescence signal generated by the magnetic protein in the droplet in response to periodic magnetic field modulation; The signal processing module is used to perform lock-in amplification on the acquired dynamic fluorescence signal, extract the signal amplitude with the same frequency as the magnetic field modulation frequency, and determine the concentration of the paramagnetic substance to be tested based on the correspondence between the signal amplitude and the preset standard curve.

2. The photonic quantum biological detection system based on a digital microfluidic platform as described in claim 1, characterized in that, The optical-magnetic co-modulation module includes a fluorescence excitation block, an objective lens, a digital microfluidic chip, an inverted fluorescence microscope, an sCMOS camera, and a light-emitting diode light source. The inverted fluorescence microscope serves as the optical imaging path and works in conjunction with the light-emitting diode light source to complete the excitation-collection process. The light-emitting diode is used to provide stable excitation light of a specific wavelength; The excitation light passes through a fluorescent excitation block and is focused onto the droplet reaction region formed on the digital microfluidic chip by an objective lens with a certain magnification. The inverted fluorescence microscope is used to collect fluorescence signals generated in the droplet reaction zone; The sCMOS camera periodically captures fluorescence signals at the location of the droplet reaction zone.

3. The photonic quantum biological detection system based on a digital microfluidic platform as described in claim 2, characterized in that, The digital microfluidic chip is used as a reaction chamber; The digital microfluidic chip includes an electrode substrate and a cover plate; The electrode substrate is provided with multiple driving electrode arrays, at least one reagent storage area, and droplet control channel; The geometric dimensions of the digital microfluidic chip are as follows: electrode size of 250μm×250μm, droplet manipulation channel height of 50-150μm, and dielectric layer thickness of 300nm.

4. The photonic quantum biological detection system based on a digital microfluidic platform as described in claim 3, characterized in that, The droplet manipulation module includes an electronic drive unit, a flexible printed circuit board, and a software interface. Engineered E. coli containing the magnetofluorescent protein MagLOV was combined with the paramagnetic metal ion gadolinium (Gd). 3+ The droplets are controlled by an electronic drive unit via a software interface to apply a voltage sequence to the electrode array of the digital microfluidic chip through a flexible printed circuit board. The principle of dielectric wetting is used to drive, transport, mix, position and refresh the droplets on the digital microfluidic chip. The droplets are manipulated from the loading area to the detection area through the electrode array, and automatically removed and replenished with fresh droplets after detection.

5. The photonic quantum biological detection system based on a digital microfluidic platform as described in claim 4, characterized in that, The optical-magnetic coordinated modulation module also includes a controllable magnetic field generation unit and a magnetic field strength adjustment unit; The controllable magnetic field generating unit is composed of an iron-core electromagnet; The magnetic field strength adjustment unit includes a signal generator, a power amplifier, and a power adapter; The signal generator outputs a pulse width modulation (PWM) signal with adjustable frequency and duty cycle. After being amplified by a power amplifier, the signal is stably powered by a power adapter to drive an iron-core electromagnet to generate a periodic modulated magnetic field.

6. The photonic quantum biological detection system based on a digital microfluidic platform as described in claim 5, characterized in that, The engineered Escherichia coli containing the MagLOV magnetic protein-expressing bacterial suspension was constructed using the pET-28a(+) vector. The MagLOV gene was inserted into the NcoI / XhoI site, and after sequencing verification, it was transformed into Escherichia coli BL21(DE3) strain.

7. The photonic quantum biological detection system based on a digital microfluidic platform as described in claim 6, characterized in that, The paramagnetic metal ions include at least one of free gadolinium ions and complexed gadolinium ions; The concentration range of the paramagnetic metal ion solution is 0.01-100 mM.

8. A photonic biodetection method based on a digital microfluidic platform, the method being used to execute the photonic biodetection system based on a digital microfluidic platform as described in any one of claims 1-7, characterized in that, include: Step S1: The engineered bacterial suspension containing MagLOV magnetic sensitive protein and the solution of the paramagnetic substance to be tested are loaded into the digital microfluidic chip to form independent engineered bacterial droplets and test substance droplets. Step S2: Apply a voltage sequence to the electrode array of the digital microfluidic chip through the digital microfluidic platform, drive the test analyte droplets and engineered bacterial droplets to merge and mix using the dielectric wetting principle, and position the mixed detection droplets to the detection area; Step S3: Apply fluorescence excitation light and periodically modulated magnetic field to the detection area, and continuously acquire dynamic fluorescence signals generated by the magnetic protein in the detection drop in response to the periodic magnetic field modulation. Step S4: The acquired dynamic fluorescence signal is amplified by lock-in processing, and the signal amplitude with the same frequency as the magnetic field modulation frequency is extracted. Based on the correspondence between the signal amplitude and the preset standard curve, the concentration of the paramagnetic substance to be tested is determined.

9. The photonic quantum biological detection method based on a digital microfluidic platform as described in claim 8, characterized in that, The method for generating the periodically modulated magnetic field in step S2 is as follows: A pulse width modulation signal is output from a signal generator, amplified by a power amplifier, and then drives an electromagnet to generate the periodic modulated magnetic field.

10. The photonic quantum biological detection method based on a digital microfluidic platform as described in claim 8, characterized in that, The specific method for continuously acquiring dynamic fluorescence signals in step S3 is as follows: The incident light is emitted by a light-emitting diode light source, passes through a fluorescence excitation block, and is focused onto the detection droplet area by an objective lens. The image sensor periodically captures the fluorescence signal at the location of the droplet reaction area.

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