Key Contributors to Signal Generation in Frequency Mixing Magnetic Detection (FMMD): An In Silico Study.

Frequency mixing magnetic detection (FMMD) is a sensitive and selective technique to detect magnetic nanoparticles (MNPs) serving as probes for binding biological targets. Its principle relies on the nonlinear magnetic relaxation dynamics of a particle ensemble interacting with a dual frequency external magnetic field. In order to increase its sensitivity, lower its limit of detection and overall improve its applicability in biosensing, matching combinations of external field parameters and internal particle properties are being sought to advance FMMD. In this study, we systematically probe the aforementioned interaction with coupled Néel-Brownian dynamic relaxation simulations to examine how key MNP properties as well as applied field parameters affect the frequency mixing signal generation. It is found that the core size of MNPs dominates their nonlinear magnetic response, with the strongest contributions from the largest particles. The drive field amplitude dominates the shape of the field-dependent response, whereas effective anisotropy and hydrodynamic size of the particles only weakly influence the signal generation in FMMD. For tailoring the MNP properties and parameters of the setup towards optimal FMMD signal generation, our findings suggest choosing large particles of core sizes dC>25 nm with narrow size distributions (σ<0.1) to minimize the required drive field amplitude. This allows potential improvements of FMMD as a stand-alone application, as well as advances in magnetic particle imaging, hyperthermia and magnetic immunoassays.

Impact of Particle Size on the Nonlinear Magnetic Response of Iron Oxide Nanoparticles during Frequency Mixing Magnetic Detection.

Magnetic nanoparticles (MNPs), particularly iron oxide nanoparticles (IONPs), play a pivotal role in biomedical applications ranging from magnetic resonance imaging (MRI) enhancement and cancer hyperthermia treatments to biosensing. This study focuses on the synthesis, characterization, and application of IONPs with two different size distributions for frequency mixing magnetic detection (FMMD), a technique that leverages the nonlinear magnetization properties of MNPs for sensitive biosensing. IONPs are synthesized through thermal decomposition and subsequent growth steps. Our findings highlight the critical influence of IONP size on the FMMD signal, demonstrating that larger particles contribute dominantly to the FMMD signal. This research advances our understanding of IONP behavior, underscoring the importance of size in their application in advanced diagnostic tools.

MPI System with Bore Sizes of 75 mm and 100 mm Using Permanent Magnets and FMMD Technique.

We present two magnetic particle imaging (MPI) systems with bore sizes of 75 mm and 100 mm, respectively, using three-dimensionally arranged permanent magnets for excitation and frequency mixing magnetic detection (FMMD) coils for detection. A rotational and a translational stage were combined to move the field free line (FFL) and acquire the MPI signal, thereby enabling simultaneous overall translation and rotational movement. With this concept, the complex coil system used in many MPI systems, with its high energy consumption to generate the drive field, can be replaced. The characteristic signal of superparamagnetic iron oxide (SPIO) nanoparticles was generated via movement of the FFL and acquired using the FMMD coil. The positions of the stages and the occurrence of the f1 + 2f2 harmonics were mapped to reconstruct the spatial location of the SPIO. Image reconstruction was performed using Radon and inverse Radon transformations. As a result, the presented method based on mechanical movement of permanent magnets can be used to measure the MPI, even for samples as large as 100 mm. Our research could pave the way for further technological developments to make the equipment human size, which is one of the ultimate goals of MPI.

A Novel Field-Free Line Generator for Mechanically Scanned Magnetic Particle Imaging.

In this study, we propose an efficient field-free line (FFL) generator for mechanically driven FFL magnetic particle imaging (MPI) applications. The novel FFL generator comprises pairs of Halbach arrays and bar magnets. The proposed design generates high-gradient FFLs with low-mass permanent magnets, realizing fine spatial resolutions in MPI. We investigate the magnetic field generated using simulations and experiments. Our results show that the FFL generator yields a high gradient of 4.76 T/m at a cylindrical field of view of 30 mm diameter and a 70 mm open bore. A spatial resolution of less than 3.5 mm was obtained in the mechanically driven FFL-MPI.

DNA Sensor for the Detection of Brucella spp. Based on Magnetic Nanoparticle Markers.

Due to the limitations of conventional Brucella detection methods, including safety concerns, long incubation times, and limited specificity, the development of a rapid, selective, and accurate technique for the early detection of Brucella in livestock animals is crucial to prevent the spread of the associated disease. In the present study, we introduce a magnetic nanoparticle marker-based biosensor using frequency mixing magnetic detection for point-of-care testing and quantification of Brucella DNA. Superparamagnetic nanoparticles were used as magnetically measured markers to selectively detect the target DNA hybridized with its complementary capture probes immobilized on a porous polyethylene filter. Experimental conditions like density and length of the probes, hybridization time and temperature, and magnetic binding specificity, sensitivity, and detection limit were investigated and optimized. Our sensor demonstrated a relatively fast detection time of approximately 10 min, with a detection limit of 55 copies (0.09 fM) when tested using DNA amplified from Brucella genetic material. In addition, the detection specificity was examined using gDNA from Brucella and other zoonotic bacteria that may coexist in the same niche, confirming the method's selectivity for Brucella DNA. Our proposed biosensor has the potential to be used for the early detection of Brucella bacteria in the field and can contribute to disease control measures.

Frequency Mixing Magnetic Detection Setup Employing Permanent Ring Magnets as a Static Offset Field Source.

Frequency mixing magnetic detection (FMMD) has been explored for its applications in fields of magnetic biosensing, multiplex detection of magnetic nanoparticles (MNP) and the determination of core size distribution of MNP samples. Such applications rely on the application of a static offset magnetic field, which is generated traditionally with an electromagnet. Such a setup requires a current source, as well as passive or active cooling strategies, which directly sets a limitation based on the portability aspect that is desired for point of care (POC) monitoring applications. In this work, a measurement head is introduced that involves the utilization of two ring-shaped permanent magnets to generate a static offset magnetic field. A steel cylinder in the ring bores homogenizes the field. By variation of the distance between the ring magnets and of the thickness of the steel cylinder, the magnitude of the magnetic field at the sample position can be adjusted. Furthermore, the measurement setup is compared to the electromagnet offset module based on measured signals and temperature behavior.

Magnetic graphene quantum dots facilitate closed-tube one-step detection of SARS-CoV-2 with ultra-low field NMR relaxometry.

The rapid and sensitive diagnosis of the highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the crucial issues at the outbreak of the ongoing global pandemic that has no valid cure. Here, we propose a SARS-CoV-2 antibody conjugated magnetic graphene quantum dots (GQDs)-based magnetic relaxation switch (MRSw) that specifically recognizes the SARS-CoV-2. The probe of MRSw can be directly mixed with the test sample in a fully sealed vial without sample pretreatment, which largely reduces the testers' risk of infection during the operation. The closed-tube one-step strategy to detect SARS-CoV-2 is developed with home-made ultra-low field nuclear magnetic resonance (ULF NMR) relaxometry working at 118 µT. The magnetic GQDs-based probe shows ultra-high sensitivity in the detection of SARS-CoV-2 due to its high magnetic relaxivity, and the limit of detection is optimized to 248 Particles mL‒1. Meanwhile, the detection time in ULF NMR system is only 2 min, which can significantly improve the efficiency of detection. In short, the magnetic GQDs-based MRSw coupled with ULF NMR can realize a rapid, safe, and sensitive detection of SARS-CoV-2.

Multiplex Detection of Magnetic Beads Using Offset Field Dependent Frequency Mixing Magnetic Detection.

Magnetic immunoassays employing Frequency Mixing Magnetic Detection (FMMD) have recently become increasingly popular for quantitative detection of various analytes. Simultaneous analysis of a sample for two or more targets is desirable in order to reduce the sample amount, save consumables, and save time. We show that different types of magnetic beads can be distinguished according to their frequency mixing response to a two-frequency magnetic excitation at different static magnetic offset fields. We recorded the offset field dependent FMMD response of two different particle types at frequencies f1 + n⋅f2, n = 1, 2, 3, 4 with f1 = 30.8 kHz and f2 = 63 Hz. Their signals were clearly distinguishable by the locations of the extremes and zeros of their responses. Binary mixtures of the two particle types were prepared with different mixing ratios. The mixture samples were analyzed by determining the best linear combination of the two pure constituents that best resembled the measured signals of the mixtures. Using a quadratic programming algorithm, the mixing ratios could be determined with an accuracy of greater than 14%. If each particle type is functionalized with a different antibody, multiplex detection of two different analytes becomes feasible.

Investigation on core-fucosylated prostate-specific antigen as a refined biomarker for differentiation of benign prostate hyperplasia and prostate cancer of different aggressiveness.

Prostate cancer represents a major cause of cancer death in men worldwide. Novel non-invasive methods are still required for differentiation of non-aggressive from aggressive tumors. Recently, changes in prostate-specific antigen glycosylation pattern, such as core-fucosylation, have been described in prostate cancer. The objective of this study was to evaluate whether the core-fucosylation determinant of serum prostate-specific antigen may serve as refined marker for differentiation between benign prostate hyperplasia and prostate cancer or identification of aggressive prostate cancer. A previously developed liquid chromatography-mass spectrometry/mass spectrometry-based strategy was used for multiplex analysis of core-fucosylated prostate-specific antigen (fuc-PSA) and total prostate-specific antigen levels in sera from 50 benign prostate hyperplasia and 100 prostate cancer patients of different aggressiveness (Gleason scores, 5-10) covering the critical gray area (2-10 ng/mL). For identification of aggressive prostate cancer, the ratio of fuc-PSA to total prostate-specific antigen (%-fuc-PSA) yielded a 5%-8% increase in the area under the curve (0.60) compared to the currently used total prostate-specific antigen (area under the curve = 0.52) and %-free prostate-specific antigen (area under the curve = 0.55) tests. However, our data showed that aggressive prostate cancer (Gleason score > 6) and non-aggressive prostate cancer (Gleason score ≤ 6) could not significantly (p-value = 0.08) be differentiated by usage of %-fuc-PSA. In addition, both non-standardized fuc-PSA and standardized %-fuc-PSA had no diagnostic value for differentiation of benign prostate hyperplasia from prostate cancer. The %-fuc-PSA serum levels could not improve the differentiation of non-aggressive and aggressive prostate cancer compared to conventional diagnostic prostate cancer markers. Still, it is unclear whether these limitations come from the biomarker, the used patient cohort, or the imprecision of the applied method itself. Therefore, %-fuc-PSA should be further investigated, especially by more precise methods whether it could be clinically used in prostate cancer diagnosis.

Multiplex Detection of Different Magnetic Beads Using Frequency Scanning in Magnetic Frequency Mixing Technique.

In modern bioanalytical methods, it is often desired to detect several targets in one sample within one measurement. Immunological methods including those that use superparamagnetic beads are an important group of techniques for these applications. The goal of this work is to investigate the feasibility of simultaneously detecting different superparamagnetic beads acting as markers using the magnetic frequency mixing technique. The frequency of the magnetic excitation field is scanned while the lower driving frequency is kept constant. Due to the particles' nonlinear magnetization, mixing frequencies are generated. To record their amplitude and phase information, a direct digitization of the pickup-coil's signal with subsequent Fast Fourier Transformation is performed. By synchronizing both magnetic fields, a stable phase information is gained. In this research, it is shown that the amplitude of the dominant mixing component is proportional to the amount of superparamagnetic beads inside a sample. Additionally, it is shown that the phase does not show this behaviour. Excitation frequency scans of different bead types were performed, showing different phases, without correlation to their diverse amplitudes. Two commercially available beads were selected and a determination of their amount in a mixture is performed as a demonstration for multiplex measurements.

3D Printed Modular Immunofiltration Columns for Frequency Mixing-Based Multiplex Magnetic Immunodetection.

For performing point-of-care molecular diagnostics, magnetic immunoassays constitute a promising alternative to established enzyme-linked immunosorbent assays (ELISA) because they are fast, robust and sensitive. Simultaneous detection of multiple biomolecular targets from one body fluid sample is desired. The aim of this work is to show that multiplex magnetic immunodetection based on magnetic frequency mixing by means of modular immunofiltration columns prepared for different targets is feasible. By calculations of the magnetic response signal, the required spacing between the modules was determined. Immunofiltration columns were manufactured by 3D printing and antibody immobilization was performed in a batch approach. It was shown experimentally that two different target molecules in a sample solution could be individually detected in a single assaying step with magnetic measurements of the corresponding immobilization filters. The arrangement order of the filters and of a negative control did not influence the results. Thus, a simple and reliable approach to multi-target magnetic immunodetection was demonstrated.

Sensor Configuration and Algorithms for Power-Line Interference Suppression in Low Field Nuclear Magnetic Resonance.

Low field (LF) nuclear magnetic resonance (NMR) shows potential advantages to study pure heteronuclear J-coupling and observe the fine structure of matter. Power-line harmonics interferences and fixed-frequency noise peaks might introduce discrete noise peaks into the LF-NMR spectrum in an open environment or in a conductively shielded room, which might disturb J-coupling spectra of matter recorded at LF. In this paper, we describe a multi-channel sensor configuration of superconducting quantum interference devices, and measure the multiple peaks of the 2,2,2-trifluoroethanol J-coupling spectrum. For the case of low signal to noise ratio (SNR) < 1, we suggest two noise suppression algorithms using discrete wavelet analysis (DWA), combined with either least squares method (LSM) or gradient descent (GD). The de-noising methods are based on spatial correlation of the interferences among the superconducting sensors, and are experimentally demonstrated. The DWA-LSM algorithm shows a significant effect in the noise reduction and recovers SNR > 1 for most of the signal peaks. The DWA-GD algorithm improves the SNR further, but takes more computational time. Depending on whether the accuracy or the speed of the de-noising process is more important in LF-NMR applications, the choice of algorithm should be made.

Prostate cancer risk regions at 8q24 and 17q24 are differentially associated with somatic TMPRSS2:ERG fusion status.

Molecular and epidemiological differences have been described between TMPRSS2:ERG fusion-positive and fusion-negative prostate cancer (PrCa). Assuming two molecularly distinct subtypes, we have examined 27 common PrCa risk variants, previously identified in genome-wide association studies, for subtype specific associations in a total of 1221 TMPRSS2:ERG phenotyped PrCa cases. In meta-analyses of a discovery set of 552 cases with TMPRSS2:ERG data and 7650 unaffected men from five centers we have found support for the hypothesis that several common risk variants are associated with one particular subtype rather than with PrCa in general. Risk variants were analyzed in case-case comparisons (296 TMPRSS2:ERG fusion-positive versus 256 fusion-negative cases) and an independent set of 669 cases with TMPRSS2:ERG data was established to replicate the top five candidates. Significant differences (P < 0.00185) between the two subtypes were observed for rs16901979 (8q24) and rs1859962 (17q24), which were enriched in TMPRSS2:ERG fusion-negative (OR = 0.53, P = 0.0007) and TMPRSS2:ERG fusion-positive PrCa (OR = 1.30, P = 0.0016), respectively. Expression quantitative trait locus analysis was performed to investigate mechanistic links between risk variants, fusion status and target gene mRNA levels. For rs1859962 at 17q24, genotype dependent expression was observed for the candidate target gene SOX9 in TMPRSS2:ERG fusion-positive PrCa, which was not evident in TMPRSS2:ERG negative tumors. The present study established evidence for the first two common PrCa risk variants differentially associated with TMPRSS2:ERG fusion status. TMPRSS2:ERG phenotyping of larger studies is required to determine comprehensive sets of variants with subtype-specific roles in PrCa.

RANKL/RANK/OPG cytokine receptor system: mRNA expression pattern in BPH, primary and metastatic prostate cancer disease.

BACKGROUND: The cytokine system RANKL (receptor activator of NF-κB ligand), its receptor RANK and the antagonist OPG (osteoprotegerin) play a critical role in bone turnover. Our investigation was conducted to describe the gene expression at primary tumour site in prostate cancer patients and correlate the results with Gleason Score and PSA level. METHODS: Seventy-one samples were obtained from prostate cancer patients at the time of radical prostatectomy and palliative prostate resection (n = 71). Patients with benign prostate hyperplasia served as controls (n = 60). We performed real-time RT-PCR after microdissection of the samples. RESULTS: The mRNA expression of RANK was highest in tumour tissue from patients with bone metastases (p < 0.001) as compared to BPH or locally confined tumours, also shown in clinical subgroups distinguished by Gleason Score (< 7 or ≥ 7, p = 0.028) or PSA level (< 10 or ≥ 10 µg/l, p = 0.004). RANKL and OPG mRNA expression was higher in tumour tissue from patients with metastatic compared to local disease. The RANKL/OPG ratio was low in normal prostate tissue and high tumours with bone metastases (p < 0.05). Expression of all three cytokines was high in BPH tissue but did not exceed as much as in the tumour tissue. CONCLUSION: We demonstrated that RANK, RANKL and OPG are directly expressed by prostate cancer cells at the primary tumour site and showed a clear correlation with Gleason Score, serum PSA level and advanced disease. In BPH, mRNA expression is also detectable, but RANK expression does not exceed as much as compared to tumour tissue.

Magnetic Detection Structure for Lab-on-Chip Applications Based on the Frequency Mixing Technique.

A magnetic frequency mixing technique with a set of miniaturized planar coils was investigated for use with a completely integrated Lab-on-Chip (LoC) pathogen sensing system. The system allows the detection and quantification of superparamagnetic beads. Additionally, in terms of magnetic nanoparticle characterization ability, the system can be used for immunoassays using the beads as markers. Analytical calculations and simulations for both excitation and pick-up coils are presented; the goal was to investigate the miniaturization of simple and cost-effective planar spiral coils. Following these calculations, a Printed Circuit Board (PCB) prototype was designed, manufactured, and tested for limit of detection, linear response, and validation of theoretical concepts. Using the magnetic frequency mixing technique, a limit of detection of 15 µg/mL of 20 nm core-sized nanoparticles was achieved without any shielding.

Synergy Effect of Combining Fluorescence and Mid Infrared Fiber Spectroscopy for Kidney Tumor Diagnostics.

Matching pairs of tumor and non-tumor kidney tissue samples of four patients were investigated ex vivo using a combination of two methods, attenuated total reflection mid infrared spectroscopy and fluorescence spectroscopy, through respectively prepared and adjusted fiber probes. In order to increase the data information content, the measurements on tissue samples in both methods were performed in the same 31 preselected positions. Multivariate data analysis revealed a synergic effect of combining the two methods for the diagnostics of kidney tumor compared to individual techniques.

Electrolyte-Gated Graphene Ambipolar Frequency Multipliers for Biochemical Sensing.

In this Letter, the ambipolar properties of an electrolyte-gated graphene field-effect transistor (GFET) have been explored to fabricate frequency-doubling biochemical sensor devices. By biasing the ambipolar GFETs in a common-source configuration, an input sinusoidal voltage at frequency f applied to the electrolyte gate can be rectified to a sinusoidal wave at frequency 2f at the drain electrode. The extraordinary high carrier mobility of graphene and the strong electrolyte gate coupling provide the graphene ambipolar frequency doubler an unprecedented unity gain, as well as a detection limit of ∼4 pM for 11-mer single strand DNA molecules in 1 mM PBS buffer solution. Combined with an improved drift characteristics and an enhanced low-frequency 1/f noise performance by sampling at doubled frequency, this good detection limit suggests the graphene ambipolar frequency doubler a highly promising biochemical sensing platform.

Vasopressin lowers renal epoxyeicosatrienoic acid levels by activating soluble epoxide hydrolase.

Activation of the thick ascending limb (TAL) Na+-K+-2Cl- cotransporter (NKCC2) by the antidiuretic hormone arginine vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here, we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5 ng/h, 3 days) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 µM 14,15-EET for 30 min reduced phosphorylation of NKCC2 at the AVP-sensitive threonine residues T96 and T101 (-66 ± 5%; P < 0.05), while 14,15-DHET had no effect. Concomitantly, isolated perfused cortical thick ascending limb pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symmetric chloride concentrations. In summary, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis.

Circulating and Tissue Expression Levels of YKL-40 in Renal Cell Cancer.

PURPOSE: Blood levels of YKL-40 are elevated in various malignancies and other inflammatory diseases. Higher YKL-40 levels have consequently been shown to correlate with poor prognosis in several cancers. We investigated the prognostic value of circulating and tissue levels of YKL-40 in renal cell cancer. MATERIALS AND METHODS: Preoperative YKL-40 serum/plasma levels were determined in 222 surgically treated patients with renal cell cancer and in 35 controls. Postoperative serum samples were analyzed in 19 of the 222 renal cell cancer cases. Gene expression levels were assessed in 101 renal cell cancer frozen tissue samples using quantitative real-time reverse transcriptase-polymerase chain reaction. Finally immunohistochemical analysis was done in 37 renal cell cancer cases to assess tissue localization of YKL-40. Results were correlated with clinicopathological and followup data. RESULTS: YKL-40 serum but not tissue gene expression levels were higher in patients with renal cell cancer compared to controls (p = 0.050). Serum YKL-40 levels significantly increased following nephrectomy (p <0.001). High circulating YKL-40 concentrations were independently associated with shorter survival in the serum and plasma cohorts. YKL-40 gene expression did not correlate with patient prognosis. CONCLUSIONS: Preoperatively elevated circulating levels of YKL-40 predict survival in patients treated with nephrectomy for renal cell cancer independently of levels determined in serum or plasma. Tumor cells do not seem to be the main source of increased serum/plasma YKL-40 levels in patients with renal cell cancer.

Dysfunction of PSA-specific CD8+ T cells in prostate cancer patients correlates with CD38 and Tim-3 expression.

The efficacy of immunotherapy in cancer patients is influenced by differences in their immune status. An evaluation of immunocompetence before therapy may help to predict therapeutic success and guide the selection of appropriate regimens. We assessed the preexisting cellular immunity against prostate-specific antigen (PSA) in untreated prostate cancer patients and healthy controls through measurement of the phenotype and function of CD8(+) T cells. Our data show that the majority of healthy men possess functional PSA-specific CD8(+) T cells in contrast to cancer patients, where <50 % showed a CD8(+) T cell response. PSA146-154-specific CD8(+) T cells of these patients had a higher expression of the activation marker CD38 and the exhaustion marker Tim-3, indicating that PSA-specific cells are exhausted. The heterogeneity of the CD8(+) T cell response against PSA in prostate cancer patients may influence their response to therapy and is a factor to be taken into account while designing and selecting treatment regimens.