Rolling Circle Amplification on a Bead: Improving the Detection Time for a Magnetic Bioassay.

Detection of pathogens has become increasingly important, especially in the face of outbreaks and epidemics all over the world. Nucleic acid detection techniques provide a solid base to detect and identify pathogens. In recent years, magnetic sensors and magnetic labels have become of more interest due to their simplicity of use, low cost, and versatility. In this work, we have used the isothermal DNA amplification technique of rolling circle amplification (RCA) in combination with oligo-functionalized magnetic nanoparticles. Detection of RCA products takes place through specific binding between magnetic nanoparticles and RCA products. Upon binding, the relaxation frequency of the nanoparticle changes. This change was measured using an AC susceptometer. We showcase that the RCA time can be reduced for a quicker assay when performing the RCA on the surface of micrometer-sized beads, which consequently increases the hydrodynamic volume of the RCA products. This, in turn, increases the Brownian relaxation frequency shift of the nanoparticles upon binding. We performed optimization work to determine the ideal quantity of micrometer-sized particles, oligo-functionalized nanoparticles, and the amplification time of the RCA. We show that the detection of 0.75 fmol of preamplification synthetic target is possible with only 20 min of amplification time. Finally, we showcase the high specificity of the assay, as the functionalized nanoparticles are unable to bind to amplified DNA that does not match their labels. Overall, this paves the way for a simple bioassay that can be used without expensive laboratory equipment for detection of pathogens in outbreak settings and clinics around the world.

Bioavailability of Celecoxib Formulated with Mesoporous Magnesium Carbonate-An In Vivo Evaluation.

An attractive approach to increase the aqueous apparent solubility of poorly soluble drugs is to formulate them in their amorphous state. In the present study, celecoxib, a poorly soluble drug, was successfully loaded into mesoporous magnesium carbonate (MMC) in its amorphous state via a solvent evaporation method. Crystallization of celecoxib was suppressed, and no reaction with the carrier was detected. The MMC formulation was evaluated in vitro and in vivo in terms of oral bioavailability. Celebra®, a commercially available formulation, was used as a reference. The two celecoxib formulations were orally administrated in male rats (average of n = 6 animals per group), and blood samples for plasma were taken from all animals at different time points after administration. There was no statistical difference (p > 0.05) in AUCinf between the two formulations. The results showed that MMC may be a promising drug delivery excipient for increasing the bioavailability of compounds with solubility-limited absorption.

Formation of Visible Aggregates between Rolling Circle Amplification Products and Magnetic Nanoparticles as a Strategy for Point-of-Care Diagnostics.

Visual detection of rolling circle amplification products (RCPs) has been achieved by specific aggregation with magnetic nanoparticles. The method presented here reliably generates aggregates in 1.5 h; these are visible to the naked eye in samples containing at least 0.4 fmol of RCPs. In addition, alternate current susceptometry and absorbance spectroscopy have also been used to quantify the amplified products. The specificity of the detection method was tested, and no non-specific aggregation was detected in samples containing up to 20 fmol of non-complementary amplified DNA. This method is a versatile tool for detecting pathogenic DNA in point-of-care diagnostics, with no readout equipment required. However, chips and automated assays can be used in conjugation with the developed method since detection and quantification can be achieved by commercially available readout instruments.

Increasing the Transport of Celecoxib over a Simulated Intestine Cell Membrane Model Using Mesoporous Magnesium Carbonate.

In the current work, mesoporous magnesium carbonate (MMC) was used to suppress crystallization of the poorly soluble drug celecoxib (CXB). This resulted in both a higher dissolution rate and supersaturation of the substance in vitro as well as an increased transfer of CXB over a Caco-2 cell membrane mimicking the membrane in the small intestine. The CXB flux over the cell membrane showed a linear behavior over the explored time period. These results indicate that MMC may be helpful in increasing the bioavailability and obtaining a continuous release of CXB, and similar substances, in vivo. Neusilin US2 was used as a reference material and showed a more rapid initial release with subsequent crystallization of the incorporated CXB in the release media. The presented results form the foundation of future development of MMC as a potential carrier for poorly soluble drugs.

Evaluating the Performance of a Magnetic Nanoparticle-Based Detection Method Using Circle-to-Circle Amplification.

This work explores several issues of importance for the development of a diagnostic method based on circle-to-circle amplification (C2CA) and oligonucleotide-functionalized magnetic nanoparticles. Firstly, the performance of the detection method was evaluated in terms of sensitivity and speed. Synthetic target sequences for Newcastle disease virus and Salmonella were used as model sequences. The sensitivity of the C2CA assay resulted in detection of 1 amol of starting DNA target with a total amplification time of 40 min for both target sequences. Secondly, the functionalization of the nanoparticles was evaluated in terms of robustness and stability. The functionalization was shown to be very robust, and the stability test showed that 92% of the oligos were still attached on the particle surface after three months of storage at 4 °C. Altogether, the results obtained in this study provide a strong foundation for the development of a quick and sensitive diagnostic assay.

Insights into the Formation of DNA-Magnetic Nanoparticle Hybrid Structures: Correlations between Morphological Characterization and Output from Magnetic Biosensor Measurements.

Understanding the binding mechanism between probe-functionalized magnetic nanoparticles (MNPs) and DNA targets or amplification products thereof is essential in the optimization of magnetic biosensors for the detection of DNA. Herein, the molecular interaction forming hybrid structures upon hybridization between DNA-functionalized magnetic nanoparticles, exhibiting Brownian relaxation, and rolling circle amplification products (DNA-coils) is investigated by the use of atomic force microscopy in a liquid environment and magnetic biosensors measuring the frequency-dependent magnetic response and the frequency-dependent modulation of light transmission. This approach reveals the qualitative and quantitative correlations between the morphological features of the hybrid structures with their magnetic response. The suppression of the high-frequency peak in the magnetic response and the appearance of a new peak at lower frequencies match the formation of larger sized assemblies upon increasing the concentration of DNA-coils. Furthermore, an increase of the DNA-coil concentration induces an increase in the number of MNPs per hybrid structure. This study provides new insights into the DNA-MNP binding mechanism, and its versatility is of considerable importance for the mechanistic characterization of other DNA-nanoparticle biosensor systems.

Effect of COD on mainstream anammox: Evaluation of process performance, granule morphology and nitrous oxide production.

The effect of COD addition into an anammox reactor was assessed during short and long term exposure. The short term exposure was assessed via batch tests and lasted 48 h. Results indicated the presence of a very active denitrifying community able to consume COD using nitrate and nitrite as electron acceptors. However, the presence of COD did not result in an increase of the ammonium concentration at the end of the tests indicating that anammox activity was not suppressed by the addition of COD. Different COD concentrations (125, 225 and 175 mg COD/L) were also added in the reactor during 3 periods within its operation (period II, III and V respectively). Long term COD addition (up to 102 d of continuous addition during period II and III) caused a decrease of the anammox activity and a shift on the microbial community, with a decrease on the anammox fraction. However, the anammox process was never lost and it fully recovered as soon as COD addition stopped. Finally, dissolved N2O was monitored under periods with and without COD addition, showing higher concentrations during transient periods from COD addition to no addition. The results of this paper provide evidence of how a long term COD exposure into an anammox reactor affect the overall nitrogen removal process, the granular structure of the anammox biomass, its microbial composition and for the first time, its N2O emissions.

Homogeneous Differential Magnetic Assay.

Assays are widely used for detection of various targets, including pathogens, drugs, and toxins. Homogeneous assays are promising for the realization of point-of-care diagnostics as they do not require separation, immobilization, or washing steps. For low concentrations of target molecules, the speed and sensitivity of homogeneous assays have hitherto been limited by slow binding kinetics, time-consuming amplification steps, and the presence of a high background signal. Here, we present a homogeneous differential magnetic assay that utilizes a differential magnetic readout that eliminates previous limitations of homogeneous assays. The assay uses a gradiometer sensor configuration combined with precise microfluidic sample handling. This enables simultaneous differential measurement of a positive test sample containing a synthesized Vibrio cholerae target and a negative control sample, which reduces the background signal and increases the readout speed. Very low concentrations of targets down to femtomolar levels are thus detectable without any additional amplification of the number of targets. Our homogeneous differential magnetic assay method opens new possibilities for rapid and highly sensitive diagnostics at the point of care.

Exploring the Use of Amine Modified Mesoporous Magnesium Carbonate for the Delivery of Salicylic Acid in Topical Formulations: In Vitro Cytotoxicity and Drug Release Studies.

Salicylic acid (SA) has for a long time been used to treat various skin disorders due to its anti-inflammatory, bacteriostatic, and antifungal properties. In the present work, mesoporous magnesium carbonate (MMC), a promising drug carrier, was modified with 3-aminopropyl-triethoxysilane to enable loading of SA. The amine modified MMC (aMMC) was successfully loaded with 8 wt.% of SA via a solvent evaporation method. SA was later completely released from the carrier in less than 15 min. Furthermore, the cytotoxicity of the functionalized material was evaluated. aMMC was found to be non-toxic for human dermal fibroblast cells with particle concentration of up to 1000 µg/mL when exposed for 48 h. The presented results form the basis of future development of aMMC as a potential carrier for SA in dermatological applications.

Development of a Sensitive Induction-Based Magnetic Nanoparticle Biodetection Method.

We developed a novel biodetection method for influenza virus based on AC magnetic susceptibility measurement techniques (the DynoMag induction technique) together with functionalized multi-core magnetic nanoparticles. The sample consisting of an incubated mixture of magnetic nanoparticles and rolling circle amplified DNA coils is injected into a tube by a peristaltic pump. The sample is moved as a plug to the two well-balanced detection coils and the dynamic magnetic moment in each position is read over a range of excitation frequencies. The time for making a complete frequency sweep over the relaxation peak is about 5 minutes (10 Hz⁻10 kHz with 20 data points). The obtained standard deviation of the magnetic signal at the relaxation frequency (around 100 Hz) is equal to about 10-5 (volume susceptibility SI units), which is in the same range obtained with the DynoMag system. The limit of detection with this method is found to be in the range of 1 pM.

Volume-amplified magnetic bioassay integrated with microfluidic sample handling and high-Tc SQUID magnetic readout.

A bioassay based on a high-Tc superconducting quantum interference device (SQUID) reading out functionalized magnetic nanoparticles (fMNPs) in a prototype microfluidic platform is presented. The target molecule recognition is based on volume amplification using padlock-probe-ligation followed by rolling circle amplification (RCA). The MNPs are functionalized with single-stranded oligonucleotides, which give a specific binding of the MNPs to the large RCA coil product, resulting in a large change in the amplitude of the imaginary part of the ac magnetic susceptibility. The RCA products from amplification of synthetic Vibrio cholera target DNA were investigated using our SQUID ac susceptibility system in microfluidic channel with an equivalent sample volume of 3 µl. From extrapolation of the linear dependence of the SQUID signal versus concentration of the RCA coils, it is found that the projected limit of detection for our system is about 1.0 × 105 RCA coils (0.2 × 10-18 mol), which is equivalent to 66 fM in the 3 µl sample volume. This ultra-high magnetic sensitivity and integration with microfluidic sample handling are critical steps towards magnetic bioassays for rapid detection of DNA and RNA targets at the point of care.

Shape anisotropy enhanced optomagnetic measurement for prostate-specific antigen detection via magnetic chain formation.

We demonstrate a homogeneous biosensor for the detection of multivalent targets by combination of magnetic nanoparticle (MNP) chains and a low-cost 405nm laser-based optomagnetic system. The MNP chains are assembled in a rotating magnetic field and stabilized by multivalent target molecules. The number of chains remaining in zero field is proportional to the target concentration, and can be quantified by optomagnetic measurements. The shape anisotropy of the MNP chains enhances the biosensor system in terms of providing efficient mixing, reduction of depletion effects (via magnetic shape anisotropy), and directly increasing the optomagnetic signal (via optical shape anisotropy). We achieve a limit of detection (LOD) of 5.5pM (0.82ng/mL) for the detection of a model multivalent molecule, biotinylated anti-streptavidin, in PBS. For the measurements of prostate-specific antigen (PSA) in 50% serum using the proposed method, we achieve an LOD of 21.6pM (0.65ng/mL) and a dynamic detection range up to 66.7nM (2µg/mL) with a sample-to-result time of approximately 20min. The performance for PSA detection therefore well meets the clinical requirements in terms of LOD (the threshold PSA level in blood is 4ng/mL) and detection range (PSA levels span from < 0.1-104ng/mL in blood), thus showing great promise for routine PSA diagnostics and for other in-situ applications.

Enhanced release of poorly water-soluble drugs from synergy between mesoporous magnesium carbonate and polymers.

The need to combat poor water solubility has increased interest in supersaturating drug delivery systems. In this study, amorphous mesoporous magnesium carbonate (MMC) was used as a drug carrier to achieve supersaturation of tolfenamic acid and rimonabant, two drug compounds with low aqueous solubility. The potential synergy between MMC and hydroxypropyl methylcellulose (HPMC), a polymer commonly included as a precipitation inhibitor in drug delivery systems, was explored with the aim of extending the time that high supersaturation levels were maintained. Release was studied under physiological conditions using USP-2 dissolution baths. A new small-scale method was developed to enable measurement of the initial drug release occurring when the MMC is immersed in the water phase. It was shown that MMC and HPMC together resulted in significant supersaturation and that the polymer enabled both the achievement of a higher API concentration and extension of the supersaturation period. The new small-scale release method showed that the release was linearly increasing with the dose and that similar release rates were observed for the two model compounds. It was hence concluded that the MMC release was diffusion limited for the compounds explored.

Optomagnetic Detection of MicroRNA Based on Duplex-Specific Nuclease-Assisted Target Recycling and Multilayer Core-Satellite Magnetic Superstructures.

Superstructural assembly of magnetic nanoparticles enables approaches to biosensing by combining specially tailored properties of superstructures and the particular advantages associated with a magnetic or optomagnetic read-out such as low background signal, easy manipulation, cost-efficiency, and potential for bioresponsive multiplexing. Herein, we demonstrate a sensitive and rapid miRNA detection method based on optomagnetic read-out, duplex-specific nuclease (DSN)-assisted target recycling, and the use of multilayer core-satellite magnetic superstructures. Triggered by the presence of target miRNA and DSN-assisted target recycling, the core-satellite magnetic superstructures release their "satellites" to the suspension, which subsequently can be quantified accurately in a low-cost and user-friendly optomagnetic setup. Target miRNAs are preserved in the cleaving reaction and can thereby trigger more cleavage and release of "satellites". For singleplex detection of let-7b, a linear detection range between 10 fM and 10 nM was observed, and a detection limit of 4.8 fM was obtained within a total assay time of 70 min. Multiplexing was achieved by releasing nanoparticles of different sizes in the presence of different miRNAs. The proposed method also has the advantages of single-nucleotide mismatch discrimination and the ability of quantification in a clinical sample matrix, thus holding great promise for miRNA routine multiplex diagnostics.

A Magnetic Nanobead-Based Read-Out Procedure for Rapid Detection of DNA Molecules.

The presented measurement and data analysis procedure reduces the read-out time for the volume-amplified magnetic nanobead detection assay from ~30 min to only 2 min, providing fast, sensitive detection of DNA molecules. The molecular detection and amplification protocol was verified using samples containing rolling circle-amplified DNA products formed from synthetic Vibrio cholerae target DNA, with a limit of detection of 5 pM. The developed read-out method could be used to rapidly identify pathogens in a variety of applications including target screening in hospitals with limited resources, in out-patient settings and in the field.

Supersaturation of poorly soluble drugs induced by mesoporous magnesium carbonate.

This work investigates whether the solubility of poorly soluble compounds can be improved by using mesoporous magnesium carbonate (MMC) as the drug delivery system. A solvent evaporation method was used to load structurally diverse model drugs (celecoxib, cinnarizine and griseofulvin) into the pores of MMC. The drug-loaded carrier system was then characterized in terms of porosity, crystallinity, and release profiles by a variety of experimental techniques, including X-ray diffraction, nitrogen adsorption analysis, differential scanning calorimetry, infrared spectroscopy, UV absorption spectroscopy, and thermogravimetric analysis. All three drugs were in a non-crystalline state after loading into the pores of MMC. The concentrations of the drugs in solution over time (a measure of the release rates from loaded MMC) were higher than the corresponding concentrations (dissolution rates) of equal amounts of the crystalline drugs. The release rates were five (celecoxib), three (cinnarizine) and two times (griseofulvin) higher than the dissolution rates of their crystalline counterparts. Supersaturation release profiles were also observed; the areas under the concentration-time curves (0-240min) were 25- (celecoxib), 5- (cinnarizine) and 2-fold (griseofulvin) greater than those of the crystalline drugs. Hence, MMC shows promise as a general drug delivery vehicle for increasing the bioavailability of compounds with dissolution rate- or solubility-limited absorption.

Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry.

Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.

Diffusion-Controlled Drug Release From the Mesoporous Magnesium Carbonate Upsalite(®).

In vitro drug release from well-defined particle-size fractions of the mesoporous magnesium carbonate material Upsalite(®) was investigated in detail using ibuprofen, a biopharmaceutics classification system class II drug, as the model compound. The weight of loaded drug corresponded to 30% of the weight of the carrier and the pores were filled to approximately 80%. The incorporated ibuprofen was found to be in an amorphous state and was physisorbed, rather than chemisorbed, to the surfaces of the pore walls. In contrast to ibuprofen in mesoporous silica, there was no detectable drug on the outer surface of the carrier particles. Two ibuprofen doses were loaded into Upsalite(®) particles with size fractions ranging from 25 µm to more than 200 µm. The initial release rate was controlled by the particle size; the dissolution rate of the loaded ibuprofen during this period was more than four times faster than that of the crystalline drug. An extended-release period of about 24 h followed the initial rapid-release period. The features of this extended-release period were dependent on the total drug concentration in the release medium. Detailed analysis of the diffusion of ibuprofen in Upsalite(®) provided the ibuprofen diffusion coefficient (9.8 × 10(-8) cm(2)/s), the constrictivity of the diffusion process (0.47) and the tortuosity of the carrier (15). This relatively high tortuosity value indicates that Upsalite(®) can be used not only to enhance the dissolution rate of poorly soluble drugs but also as a carrier in sustained-release applications by using larger particle sizes or even pellets of the material.

Novel readout method for molecular diagnostic assays based on optical measurements of magnetic nanobead dynamics.

We demonstrate detection of DNA coils formed from a Vibrio cholerae DNA target at picomolar concentrations using a novel optomagnetic approach exploiting the dynamic behavior and optical anisotropy of magnetic nanobead (MNB) assemblies. We establish that the complex second harmonic optical transmission spectra of MNB suspensions measured upon application of a weak uniaxial AC magnetic field correlate well with the rotation dynamics of the individual MNBs. Adding a target analyte to the solution leads to the formation of permanent MNB clusters, namely, to the suppression of the dynamic MNB behavior. We prove that the optical transmission spectra are highly sensitive to the formation of permanent MNB clusters and, thereby to the target analyte concentration. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition and isothermal rolling circle amplification and benchmark against a commercial equipment. The results demonstrate the fast optomagnetic readout of rolling circle products from bacterial DNA utilizing the dynamic properties of MNBs in a miniaturized and low-cost platform requiring only a transparent window in the chip.

Quantification of rolling circle amplified DNA using magnetic nanobeads and a Blu-ray optical pick-up unit.

We present the first implementation of a Blu-ray optical pickup unit (OPU) for the high-performance low-cost readout of a homogeneous assay in a multichamber microfluidic disc with a chamber thickness of 600 µm. The assay relies on optical measurements of the dynamics of magnetic nanobeads in an oscillating magnetic field applied along the light propagation direction. The laser light provided by the OPU is transmitted through the sample chamber and reflected back onto the photo detector array of the OPU via a mirror. Spectra of the 2nd harmonic photo detector signal vs. the frequency of the applied magnetic field show a characteristic peak due to freely rotating magnetic nanobeads. Beads bound to ~1 µm coils of DNA formed off-chip by padlock probe recognition and rolling circle amplification show a different dynamics and the intensity of the characteristic peak decreases. We have determined the optimum magnetic bead concentration to 0.1mg/mL and have measured the response vs. concentration of DNA coils formed from Escherichia Coli. We have found a limit of detection of 10 pM and a dynamic range of about two orders of magnitude, which is comparable to the performance obtained using costly and bulky laboratory equipment. The presented device leverages on the advanced but low-cost technology of Blu-ray OPUs to provide a low-cost and high-performance magnetic bead-based readout of homogeneous bioassays. The device is highly flexible and we have demonstrated its use on microfluidic chambers in a disc with a thickness compatible with current optical media mass-production facilities.