Supramolecular Mimotope Peptide Nanofibers Promote Antibody-Ligand Polyvalent and Instantaneous Recognition for Biopharmaceutical Analysis.

Peptide-based supramolecules exhibit great potential in various fields due to their improved target recognition ability and versatile functions. However, they still suffer from numerous challenges for the biopharmaceutical analysis, including poor self-assembly ability, undesirable ligand-antibody binding rates, and formidable target binding barriers caused by ligand crowding. To tackle these issues, a "polyvalent recognition" strategy employing the CD20 mimotope peptide derivative NBD-FFVLR-GS-WPRWLEN (acting on the CDR domains of rituximab) was proposed to develop supramolecular nanofibers for target antibody recognition. These nanofibers exhibited rapid self-assembly within only 1 min and robust stability. Their binding affinity (179 nM) for rituximab surpassed that of the monomeric peptide (7 µM) by over 38-fold, highlighting that high ligand density and potential polyvalent recognition can efficiently overcome the target binding barriers of traditional supramolecules. Moreover, these nanofibers exhibited an amazing "instantaneous capture" rate (within 15 s), a high recovery (93 ± 3%), and good specificity for the target antibody. High-efficiency enrichment of rituximab was achieved from cell culture medium with good recovery and reproducibility. Intriguingly, these peptide nanofibers combined with bottom-up proteomics were successful in tracking the deamidation of asparagine 55 (from 10 to 16%) on the rituximab heavy chain after 21 day incubation in human serum. In summary, this study may open up an avenue for the development of versatile mimotope peptide supramolecules for biorecognition and bioanalysis of biopharmaceuticals.

Construction of a Novel Semiautomated Electrochemical Sensor Array Platform and Its Application in Multiplexed Monitoring of Antibiotic Therapy.

At present, traditional analytical methods suffer from issues such as complex operation, expensive equipment, and a lengthy testing time. Electrochemical sensors have shown great advantages and application potential as an alternative solution. In this study, we proposed a novel semiautomated electrochemical sensor array (SAESA) platform. The sensor array was fabricated using screen-printed technology with a tubular design where all electrodes were printed on the inner wall. The integration of the tubular sensor array with a pipet allows for a semiautomated process including sampling and rinsing, which simplifies operation and reduces overall time. Each working electrode in the tubular sensor array underwent distinct decoration to get specific sensing responses toward the target analytes in a mixture environment (e.g., blood samples). To demonstrate the applicability of the developed sensing platform for simultaneous multianalyte detection, we chose antibiotic treatment for inflammatory infection as a model scenario and continuously measured three biomarkers, namely, tigecycline (TGC), procalcitonin (PCT), and alanine aminotransferase (ALT). The detection limits were 0.3 µM, 0.3 ng/L, and 2.76 U/L, respectively. The developed semiautomated electrochemical sensor array exhibits characteristics such as rapid and simple operation, portability, good selectivity, and excellent stability.

Mimotope peptide modified pompon mum-like magnetic microparticles for precise recognition, capture and biotransformation analysis of rituximab in biological fluids.

Due to low immobilized ligand density, limited binding capacity, and severe interference from serum proteins, developing ideal peptide-based biomaterials for precise recognition and in vivo analysis of biopharmaceuticals remains a huge challenge. In this study, mimotope peptide modified pompon mum-like biomimetic magnetic microparticles (MMPs, 3.8 µm) that mimic the specific functionalities of CD20 on malignant B cells were developed for the first time. Benefit from the numerous ligand binding sites (Ni2+) on the pompon mum-like MMPs, these novel materials achieved ≥10 times higher peptide ligand densities (>2300 mg/g) and antibody binding capacities (1380 mg/g) compared to previous reported biomaterials. Leveraging the high specificity of the mimotope peptide, rituximab can be precisely recognized and enriched from cell culture media or serum samples. We also established an LC‒MS/MS method using the MMPs for tracking rituximab biotransformation in patient serum. Intriguingly, deamidation of Asn55 and Asn33, as well as oxidation of Met81 and Met34 were observed at the key complementarity determining regions of rituximab, which could potentially influence antibody function and require careful monitoring. Overall, these versatile biomimetic MMPs demonstrate superior recognition and enrichment capabilities for target antibodies, offering interesting possibilities for biotransformation analysis of biopharmaceuticals in patient serum.

Biomimetic affinity membrane roll column for rapid purification of C-reactive protein.

To in-depth explore the action mechanism of C-reactive protein (CRP) and precisely study its signaling pathways, it is essential to acquire high-purity CRP while preserving its intact structure and functionality. In this study, we propose and fabricate a high-density 2-methacryloyloxyethyl phosphorylcholine (MPC)-modified membrane roll column (MPC-MRC) using a surface-initiated atom transfer radical polymerization (SI-ATRP) approach, which can overcome these limitations (long incubation time and low adsorption capacity) of conventional enrichment materials. The MPC-MRC incorporates a high-density 2-hydroxyethyl methacrylate polymer brush to prevent non-specific protein adsorption and multiple MPC polymer brush layers for high-performance enrichment of CRP in the company of calcium ions. Furthermore, the MPC-MRC exhibits high permeability, hydrophilicity, and mechanical strength. Compared to previous technologies, this novel material demonstrates significantly higher CRP binding capacity (310.3 mg/g), shorter processing time (only 15 min), and lower cost (only 12 USD/column). Notably, the MPC-MRC enables fast and effective purification of CRP from both human and rat serum, exhibiting good selectivity, recovery (> 91.3 %), and purity (> 95.2 %). Thus, this proposed purification approach based on MPC-MRC holds great potential for target protein enrichment from complex samples, as well as facilitating in-depth studies of its biological functions.

A chimeric hairpin DNA aptamer-based biosensor for monitoring the therapeutic drug bevacizumab.

The accurate and rapid detection of specific antibodies in blood is very important for efficient diagnosis and precise treatment. Conventional methods often suffer from time-consuming operations and/or a narrow detection range. In this work, for the rapid determination of bevacizumab in plasma, a series of chimeric hairpin DNA aptamer-based probes were designed by the modification, labeling and theoretical computation of an original aptamer. Then, the dissociation constant of the modified hairpin DNA to bevacizumab was measured and screened using microscale thermophoresis. The best chimeric hairpin DNA aptamer-based probe was then selected, and a one-step platform for the rapid determination of bevacizumab was constructed. This strategy has the advantages of being simple, fast and label-free. Because of the design and screening of the hairpin DNA, as well as the optimization of the concentration and electrochemical parameters, a low detection limit of 0.37 pM (0.054 ng mL-1) with a wide linear range (1 pM-1 µM) was obtained. Finally, the rationally constructed biosensor was successfully applied to the determination of bevacizumab in spiked samples, and it showed good accuracy and precision. This method is expected to truly realize accurate and rapid detection of bevacizumab and provides a new idea for the precise treatment of diseases.

Biopanning of specific peptide for SARS-CoV-2 nucleocapsid protein and enzyme-linked immunosorbent assay-based antigen assay.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide which triggered serious public health issues. The search for rapid and accurate diagnosis, effective prevention, and treatment is urgent. The nucleocapsid protein (NP) of SARS-CoV-2 is one of the main structural proteins expressed and most abundant in the virus, and is considered a diagnostic marker for the accurate and sensitive detection of SARS-CoV-2. Herein, we report the screening of specific peptides from the pIII phage library that bind to SARS-CoV-2 NP. The phage monoclone expressing cyclic peptide N1 (peptide sequence, ACGTKPTKFC, with C&C bridged by disulfide bonding) specifically recognizes SARS-CoV-2 NP. Molecular docking studies reveal that the identified peptide is bound to the "pocket" region on the SARS-CoV-2 NP N-terminal domain mainly by forming a hydrogen bonding network and through hydrophobic interaction. Peptide N1 with the C-terminal linker was synthesized as the capture probe for SARS-CoV-2 NP in ELISA. The peptide-based ELISA was capable of assaying SARS-CoV-2 NP at concentrations as low as 61 pg/mL (∼1.2 pM). Furthermore, the as-proposed method could detect the SARS-CoV-2 virus at limits as low as 50 TCID50 (median tissue culture infective dose)/mL. This study demonstrates that selected peptides are powerful biomolecular tools for SARS-CoV-2 detection, providing a new and inexpensive method of rapidly screening infections as well as rapidly diagnosing coronavirus disease 2019 patients.

Inside-Out Oriented Choline Phosphate-Based Biomimetic Magnetic Nanomaterials for Precise Recognition and Analysis of C-Reactive Protein.

Phospholipid-based materials exhibit great application potential in the fields of chemistry, biology, and pharmaceutical sciences. In this study, an inside-out oriented choline phosphate molecule, 2-{2-(methacryloyloxy)ethyldimethylammonium}ethyl n-butyl phosphate (MBP), was proposed and verified as a novel ligand of C-reactive protein (CRP) to enrich the functionality of these materials. Compared with phosphorylcholine (PC)-CRP interactions, the binding between MBP and CRP was not affected by the reverse position of phosphate and choline groups and even found more abundant binding sites. Thus, high-density MBP-grafted biomimetic magnetic nanomaterials (MBP-MNPs) were fabricated by reversible addition-fragmentation chain transfer polymerization based on thiol-ene click chemistry. The novel materials exhibited multifunctional applications for CRP including purification and ultrasensitive detection. On the one hand, higher specificity, recovery (90%), purity (95%), and static binding capacity (198.14 mg/g) for CRP were achieved on the novel materials in comparison with traditional PC-based materials, and the enriched CRP from patient serum can maintain its structural integrity and bioactivity. On the other hand, the CRP detection method combining G-quadruplex and thioflavin T developed with MBP-MNPs showed a lower detection limit (10 pM) and wider linear range (0.1-50 nM) than most PC-functionalized analytical platforms. Therefore, the inside-out oriented choline phosphate can not only precisely recognize CRP but also be combined with biomimetic nanomaterials to provide high application potential.

Tetrapeptide-based mimotope affinity monolith for the enrichment and analysis of anti-HER2 antibody and antibody-drug conjugate.

Selective enrichment and analysis of therapeutic antibodies in biological fluids are crucial for the development of biopharmaceuticals. Recently, peptide-based affinity chromatography has exhibited fascinating prospects for antibody enrichment due to the high affinity and specificity of small peptides. However, the post-modification approach of peptide ligands on the material surface is complicated and time-consuming. In this study, a methacrylate modified tetrapeptide (m-EDPW) was firstly demonstrated as the affinity ligand of trastuzumab (Kd = 1.91 ± 1.81 µM). Next, the m-EDPW based affinity monolith was prepared using a facile one-step polymerization method, which could overcome the drawbacks of traditional post-modification preparation strategies. Based on the monolith as described above, a simple enrichment approach was developed under the optimal washing and elution conditions. Based on the excellent properties, such as high porosity (53.09%), weak electrostatic interaction and suitable affinity (1.00 ± 2.14 µM for anti-HER2 ADC), this novel monolith exhibited good specificity and recovery for antibodies (91.6% for trastuzumab, 98.37% for anti-HER2 ADC), and low nonspecific adsorption for human serum albumin (DBC10% = 0.5 mg/g polymer). Particularly, this material was successfully applied to enrich trastuzumab and its related antibody-drug conjugate (ADC) from different cell culture medias. The dynamic tracking analysis of ADC in the critical quality attributes (e.g., charge variants, drug to antibody ratio and subunit conjugation ratio) was also achieved by combining the enrichment approach, capillary electrophoresis or reversed phase liquid chromatography. In summary, the exploited peptide-based mimotope affinity materials showed a great potential for the application in biopharmaceutical analysis.

On-site Simultaneous Determination of Neonicotinoids, Carbamates, and Phenyl Pyrazole Insecticides in Vegetables by QuEChERS Extraction on Nitrogen and Sulfur co-doped Carbon Dots and Portable Mass Spectrometry.

In food safety monitoring, on-site and simultaneous detection of a variety of insecticides with different concentrations in the same matrix is necessary. However, the task remains challenging. In this study, a novel nitrogen and sulfur co-doped carbon dot (N, S-CD) was synthesized and used as a QuEChERS clean-up reagent to reduce matrix interferences in the determination of insecticides in vegetables. In addition, a portable mass spectrometer (µ-MS) was employed, without chromatography separation, to directly determine neonicotinoids, carbamates, and benzopyrazole insecticides (with acetamiprid, imidacloprid, thiamethoxam, fipronil, and carbofuran as models) in the pretreated samples. The N,S-CD µ-MS method exhibited effective clean-up performance with satisfactory matrix effects between -15.2% and 15.7%. The recoveries of spiked vegetable samples ranged from 82.2% to 109.7% for the five target insecticides, and the relative standard deviations (RSDs) ranged from 3.8% to 16.5%. The linear ranges were from 2.0 to 5.0 ng/g, with low detection limits (LOD) from 0.5 to 1.0 ng/g. Moreover, the total pretreatment and detection time was within 20 min. Thus, the incorporation of N,S-CD with QuEChERS extraction, together with the portable µ-MS system, could be a promising and feasible strategy for on-site, rapid, and simultaneous detection of various insecticides in vegetables.

Ovulation induction drug and ovarian cancer: an updated systematic review and meta-analysis.

OBJECTIVE: To explore the association between ovulation induction drugs and ovarian cancer. DESIGN: Systematic review and meta-analysis. SETTING: Not applicable. PATIENT(S): Women without ovarian cancer who ever or never underwent ovarian induction. INTERVENTION(S): An extensive electronic search of the following databases was performed: PubMed, EMBASE, MEDLINE, Google Scholar, Cochrane Library and CNKI, from inception until January 2022. A total of 34 studies fulfilled our inclusion criteria and were included in the final meta-analysis. The odds ratio (OR) and random-effects model were used to estimate the pooled effects. The Newcastle-Ottawa Scale was used to assess the quality of included studies. Funnel plots and Egger tests were used to assess publication bias. MAIN OUTCOMES: New diagnosed borderline ovarian tumor (BOT) and invasive ovarian cancer (IOC) between ovulation induction (OI) group and control (CT) group considering fertility outcome, OI cycles and specific OI drugs. RESULTS: Primarily, there was no significant difference in the incidence of IOC and BOT between the OI and CT groups. Secondly, OI treatment did not increase the risk of IOC and BOT in the multiparous women, nor did it increase the risk of IOC in the nulliparous women. However, the risk of BOT appeared to be higher in nulliparous women treated with OI treatment. Thirdly, among women exposed to OI, the risk of IOC and BOT was higher in nulliparous women than in multiparous women. Fourthly, the risk of IOC did not increase with increasing OI cycles. Lastly, exposure to specific OI drugs also did not contribute to the risk of IOC and BOT. CONCLUSION: Overall, OI treatment did not increase the risk of IOC and BOT in most women, regardless of OI drug type and OI cycle. However, nulliparous women treated with OI showed a higher risk of ovarian cancer, necessitating their rigorous monitoring and ongoing follow-up.

Portable ATP bioluminescence sensor with high specificity for live Escherichia coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation.

Live foodborne pathogens proliferate rapidly and do great harm to human health, which requires appropriate methods to supervise. In this work, a portable adenosine triphosphate (ATP) bioluminescence sensor with high specificity for live E. coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation was developed. In brief, the selected phages were directionally immobilized on the poly(diallyldimethylammonium chloride)-modified gold stir bar as the bioreceptor. Following the simple stir bar absorptive extraction and bio-proliferation in the Luria-Bertani medium, the number of captured E. coli O157:H7 exploded. Finally, it was quantified by a portable ATP bioluminescence sensor. Benefitting from the high specificity of phage and simple signal dual-amplification strategy, the proposed biosensor achieved the recognition of live bacteria at strain level with superior sensitivity. Also, the portable signal readout made it suitable for on-site detection. Under optimal conditions, this bioassay provided a detectable range of 102-107 CFU mL-1 with a low detection limit of 30 CFU mL-1 within 30 min. The detection results for real samples demonstrated that there were no differences between the assay and the plate counting method, while the detection time was largely shortened. Furthermore, the assay gives a novel path for the point-of-care test (POCT) of live E. coli strain, which is promising to be extended to other virulent strains measurement with corresponding phages.

Peptide nano 'bead-grafting' for SDT-facilitated immune checkpoints blocking.

Combination therapies based on immune checkpoint blockade (ICB) are currently the mainstay of cancer treatment, in which the synergetic delivery of multiple drugs is the essential step. Although nanoparticle drugs (NPDs) show satisfactory anticancer effects, the promotion of active co-delivery of NPDs is premature, since the processes are usually difficult to predict and control. Targeting peptide self-assemblies have been widely used as carriers for small-molecular drugs, but remain elusive for NPDs. We describe here peptide-based nano 'bead-grafting' for the active delivery of quantum-dot NPDs through a co-assembly method. Based on a 'de novo' design, we used a 'one-bead-one-compound (OBOC)' combinatorial chemical screening method to select a peptide RT with high affinity for the immune checkpoint CD47, which could also form biocompatible nanofibers and efficiently trap Ag2S quantum dots along the self-assembly path. This system can combine ICB therapy and sonodynamic therapy (SDT) to effectively inhibit tumor growth. Moreover, the tumor antigen produced by SDT can activate the adaptive immune system, which enhances the anti-tumor immune response of the ICB and shows efficient inhibition of both primary and distant tumors. This study provides a new strategy for the active control and delivery of NPDs and a new option for ICB therapy with immune checkpoints that are highly susceptible to systemic side effects.

Rapid fabrication of zwitterionic sulfobetaine vinylimidazole-based monoliths via photoinitiated copolymerization for hydrophilic interaction chromatography.

Zwitterionic sulfobetaine-based monolithic stationary phases have attracted increasing attention for their use in hydrophilic interaction chromatography. In this study, a novel hydrophilic polymeric monolith was fabricated through photo-initiated copolymerization of 3-(3-vinyl-1-imidazolio)-1-propanesulfonate (SBVI) with pentaerythritol triacrylate using methanol and tetrahydrofuran as the porogenic system. Notably, the duration for the preparation of this novel monolith was as little as 5 min, which was significantly shorter than that required for previously reported sulfobetaine-based monoliths prepared via conventional thermally initiated copolymerization. Moreover, these monoliths showed good morphology, permeability, porosity (62.4%), mechanical strength (over 15 MPa), column efficiency (51,230 plates/m), and reproducibility (relative standard deviations for all analytes were lower than 4.6%). Mechanistic studies indicated that strong hydrophilic and negative electrostatic interactions might be responsible for the retention of polar analytes on the zwitterionic SBVI-based monolith. In particular, the resulting monolith exhibited good anti-protein adhesion ability and low nonspecific protein adsorption. These excellent features seem to favor its application in bioanalysis. Therefore, the novel zwitterionic sulfobetaine-based monolith was successfully employed for the highly selective separation of small bioactive compounds and the efficient enrichment of N-glycopeptides from complex samples. In this study, we prepared a novel zwitterionic sulfobetaine-based monolith with good performance and developed a simpler and faster method for preparation of zwitterionic monoliths.

Combining portable mass spectrometer with bamboo stir bar sorptive extraction for the on-site detection of malachite green, crystal violet and their metabolites in fishes.

In this work, a disposable and inexpensive bamboo stir bar containing an organic membrane was constructed to perform stir bar sorptive extraction (SBSE), followed by portable mass spectrometry to achieve on-site detection of four residual drugs (malachite green, crystal violet and their metabolites) in fishes. The entire method uses only microliter quantities of organic solvents, enabling environmentally friendly pretreatment. The portable mass spectrometer can simultaneously detect four target analytes in a sample in approximately ten seconds. Under the optimal conditions, the proposed method was successfully applied to simultaneously detect four drugs in fish samples with detection limits of 0.16-0.59 µg/kg. The spiked recoveries for mandarin fish and lateolabrax maculatus were 74.5-96.5%, with relative standard deviations (RSD) of 4.4-16%. In addition, the matrix effects of the four analytical targets in the method were experimentally verified to range from 7.30% to 20.62%. The method can potentially be extended to the on-site detection of other veterinary drug residues in foods.

In vitro/in vivo degradation analysis of trastuzumab by combining specific capture on HER2 mimotope peptide modified material and LC-QTOF-MS.

Degradation analysis of therapeutic mAb is of high interest for critical quality attributes assessment and biotransformation studies. However, some obstacles, including low in vivo concentrations of mAb and complex biological matrices containing IgGs, could seriously interfere with mAb bioanalysis. In this study, a bioanalytical platform was developed for studying in vitro/in vivo modifications of trastuzumab, in which specific capture on mimotope peptide modified material was combined with trypsin digestion and LC-QTOF-MS analysis. It is worth noting that this material exhibits high specificity, suitable dynamic binding capacity, very little non-specific protein adsorption, and thus provides good enrichment and quantification performances for trastuzumab from patient serums. In particular, this bioanalytical platform was successfully applied to the dynamic monitoring of modifications of trastuzumab, such as deamidation, isomerization, oxidation and cyclization. Except for the faster deamidation of LC-Asn-30 and HC-Asn-387/392/393 under serum incubation, similar degradation trends for other sites were observed in phosphate buffer and spiked serum. Differences of peptide modification degrees of trastuzumab in patient serums were also observed. The novel platform exhibited superior specificity than Protein A/G/L based analytical methods, lower cost and higher stability than antigen or anti-idiotypic antibody based analytical methods, ensuring the evaluation of modification sites.

Phosphatidylethanolamine functionalized biomimetic monolith for immobilized artificial membrane chromatography.

In this research, a new phospholipid based monolith was fabricated by in situ co-polymerization of 1-dodecanoyl-2-(11-methacrylamidoundecanoyl)-sn-glycero-3-phosphoethanolamine and ethylene dimethacrylate to mimick bio-membrane environment. Excellent physicochemical properties of this novel monolith that were achieved included column efficiency, stability, and permeability. Moreover, the biomimetic monolith showed outstanding separation capability for a series of intact proteins and small molecules. In particular, it exhibited good potential as an alternative to the commercial immobilized artificial membrane (IAM) column (IAM.PC.DD2) for studying drug-membrane interactions. This study not only enriched the types of IAM stationary phases, but also provided a simple model for the prediction of phosphatidylethanolamine related properties of drug candidates.

Fluorescent aptasensor for detection of live foodborne pathogens based on multicolor perovskite-quantum-dot-encoded DNA probes and dual-stirring-bar-assisted signal amplification.

In this study, a fluorescent (FL) aptasensor was developed for on-site detection of live Salmonella typhimurium (S.T.) and Vibrio parahaemolyticus (V.P.). Complementary DNA (cDNA) of aptamer (Apt)-functionalized multicolor polyhedral oligomeric silsesquioxane-perovskite quantum dots (cDNA-POSS-PQDs) were used as encoded probes and combined with dual-stirring-bar-assisted signal amplification for pathogen quantification. In this system, bar 1 was labeled with the S.T. and V.P. Apts, and then bar 2 was functionalized with cDNA-POSS-PQDs. When S.T. and V.P. were introduced, pathogen-Apt complexes would form and be released into the supernatant from bar 1. Under agitation, the two complexes reached bar 2 and subsequently reacted with cDNA-POSS-PQDs, which were immobilized on MXene. Then, the encoded probes would be detached from bar 2 to generate FL signals in the supernatant. Notably, the pathogens can resume their free state and initiate next cycle. They swim between the two bars, and the FL signals can be gradually enhanced to maximum after several cycles. The FL signals from released encoded probes can be used to detect the analytes. In particular, live pathogens can be distinguished from dead ones by using an assay. The detection limits and linear range for S.T. and V.P. were 30 and 10 CFU/mL and 102-106 CFU/mL, respectively. Therefore, this assay has broad application potential for simultaneous on-site detection of various live pathogenic bacteria in water.

On-Site Biolayer Interferometry-Based Biosensing of Carbamazepine in Whole Blood of Epileptic Patients.

On-site monitoring of carbamazepine (CBZ) that allows rapid, sensitive, automatic, and high-throughput detection directly from whole blood is of urgent demand in current clinical practice for precision medicine. Herein, we developed two types (being indirect vs. direct) of fiber-optic biolayer interferometry (FO-BLI) biosensors for on-site CBZ monitoring. The indirect FO-BLI biosensor preincubated samples with monoclonal antibodies towards CBZ (MA-CBZ), and the mixture competes with immobilized CBZ to bind towards MA-CBZ. The direct FO-BLI biosensor used sample CBZ and CBZ-horseradish peroxidase (CBZ-HRP) conjugate to directly compete for binding with immobilized MA-CBZ, followed by a metal precipitate 3,3'-diaminobenzidine to amplify the signals. Indirect FO-BLI detected CBZ within its therapeutic range and was regenerated up to 12 times with negligible baseline drift, but reported results in 25 min. However, Direct FO-BLI achieved CBZ detection in approximately 7.5 min, down to as low as 10 ng/mL, with good accuracy, specificity and negligible matric interference using a high-salt buffer. Validation of Direct FO-BLI using six paired sera and whole blood from epileptic patients showed excellent agreement with ultra-performance liquid chromatography. Being automated and able to achieve high throughput, Direct FO-BLI proved itself to be more effective for integration into the clinic by delivering CBZ values from whole blood within minutes.

A systematic investigation of the effect of sample solvent on peak shape in nano- and microflow hydrophilic interaction liquid chromatography columns.

A frequently encountered problem in the practical application of nano- and microflow hydrophilic interaction chromatography (HILIC) columns is the distortion of peak shapes arising from a mismatch between the sample solvent and the mobile phase. An unmatched or improperly matched sample solvent can distort the peak shape of analytes and influence their retention times, thereby affecting the quality of the resulting chromatogram. In this work, the effect of sample solvent composition (mixtures of acetonitrile, water, methanol and isopropanol in different ratios) and injection volume (20-100 nL) was systematically investigated using a selection of neutral and charged compounds on a series of zwitterionic and charged small I.D. (0.1-0.3 mm) HILIC columns. For retained compounds, pure ACN was demonstrated to be the best sample solvent to obtain narrow peaks, while for compounds that eluted very close to the solvent peak, the peak shape was distorted when the sample solvent consisted of pure ACN. A highly aqueous sample solvent, which interferes with the partitioning of polar analytes into the stationary phase, was demonstrated to be detrimental for the peak shape of retained neutral compounds, while for unretained compounds that do not or hardly interact with the stationary phase, a high amount of water in the sample solvent was not problematic. For charged compounds, water in the sample solvent favored the electrostatic attraction with the stationary phase. Therefore, the retention time of charged analytes was shown to increase with increasing water content in the sample solvent. Even when a large amount of water was present in the sample solvent, the peak shapes of these compounds were still acceptable. For highly polar compounds with a limited solubility in aqueous sample solvents, it was found that a mixture of ACN and MeOH or IPA is a good alternative.

Development of zirconium modified adenosine triphosphate functionalized monolith for specific enrichment of N-glycans.

In this study, to selectively enrich N-glycans from complex biological samples, a novel Zr(IV) modified adenosine triphosphate (Zr(IV)-ATP) functionalized monolith was prepared through a facile approach. Well-defined macroporous structure was observed in the ATP functionalized monolith, which allows rapid mass transfer under low backpressure and is beneficial for the enrichment of N-glycans. After being modified with Zr(IV), the resulting Zr(IV)-ATP functionalized monolith could selectively capture N-glycans through the specific interactions between the sulfonate groups of 1-aminopyrene-3,6,8-trisulfonic acid (APTS) labeled N-glycans and Zr(IV). An APTS labeled maltooligosaccharide ladder was used to optimize the enrichment conditions for APTS labeled N-glycans, and capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detector was employed to evaluate the enrichment efficiency. The results show that the APTS labeled maltooligosaccharides could be enriched under the selected conditions and the signal amplify factors of the maltooligosaccharides were between 7.4 and 19.5 with RSDs for reproducibility from 4.0% to 8.3% (n = 3). Finally, the proposed method was successfully used for the enrichment and detection of N-glycans released from Ribonuclease B.