Incubation In Solution Research Articles

Comparison of Incubation Solutions Prior to the Purification of Porcine Islet Cells.

For pancreatic islet transplantation, one of the most important steps of islet isolation is islet purification. The most common method of islet purification is density gradient centrifugation because there are differences in density between islets and acinar tissue. However, the density of islets/acinar tissue depends on several conditions, such as the incubation time before purification and the osmolality of the preincubation solution. In this study, we evaluated the impact of using two different preincubation solutions before purification. We used the University of Wisconsin (UW) solution and a new preservation solution (HN-1), which we recently developed. There were no significant differences between the two solutions in terms of the islet yield, rate of viability, and purity or stimulation index after purification. There were also no differences in the attainability and suitability of posttransplantation normoglycemia. Our study shows that the HN-1 solution is equivalent to the UW solution for preincubation before islet purification.

GW24-e0236 Effects of 5-aminolevulinic acid mediated sonodynamic therapy on macrophages

ObjectivesInflammatory cells exhibit elevated level of protoporphyrin IX (PpIX) after the administration of 5-aminolevulinic acid (ALA). This study aims to investigate the sonodynamic effects of ALA derived PpIX (ALA-PpIX) on...

Amyloid-β peptide (1–42) aggregation induced by copper ions under acidic conditions

It is well known that the aggregation of amyloid-β peptide (Aβ) induced by Cu²⁺ is related to incubation time, solution pH, and temperature. In this work, the aggregation of Aβ₁₋₄₂ in the presence of Cu²⁺ under acidic conditions was studied at different incubation time and temperature (e.g. 25 and 37°C). Incubation temperature, pH, and the presence of Cu²⁺ in Aβ solution were confirmed to alter the morphology of aggregation (fibrils or amorphous aggregates), and the morphology is pivotal for Aβ neurotoxicity and Alzheimer disease (AD) development. The results of atomic force microscopy (AFM) indicated that the formation of Aβ fibrous morphology is preferred at lower pH, but Cu²⁺ induced the formation of amorphous aggregates. The aggregation rate of Aβ was increased with the elevation of temperature. These results were further confirmed by fluorescence spectroscopy and circular dichroism spectroscopy and it was found that the formation of β-sheet structure was inhibited by Cu²⁺ binding to Aβ. The result was consistent with AFM observation and the fibrillation process was restrained. We believe that the local charge state in hydrophilic domain of Aβ may play a dominant role in the aggregate morphology due to the strong steric hindrance. This research will be valuable for understanding of Aβ toxicity in AD.

Biomimetic composite scaffolds based mineralization of hydroxyapatite on electrospun calcium-containing poly(vinyl alcohol) nanofibers

Nanocomposite materials consisting of polymer matrix and inorganic salts in the form of nanocrystals of hydroxyapatite (HA) are regarded as superior candidates for bone treatment. A biomimetic nanocomposite scaffold with HA formation on the electrospun poly(vinyl alcohol) (PVA) nanofibrous structure by employing a Ca–P alternate soaking method was developed in this work. The calcium-containing PVA nanofibers were prepared by adding calcium nitrate to the starting solution prior to electrospinning, and then mineralized by Ca–P treatment in incubation solution. With this rapid and effective procedure, a continuous biomimetic crystalline HA layer could be formed successfully without the need of a prior chemical modification of the substrate surface under very mild reaction conditions. Moreover, the HA formed with a relatively accelerated growth had a carbonated and poor crystalline structure, resembling biological apatite in the bone mineral. The introduction of calcium ions in nanofibers by electrospinning was a favorable approach to induce the deposition of calcium phosphate and improve the distribution, nucleation, and growth of crystalline HA layer on nanofibrous scaffolds. Bioactivity tests revealed that these mineralized PVA/HA composite scaffolds improved the biocompatibility. The porous polymer/HA composite scaffolds produced in the present study might have potential applications in bone tissue engineering.

Selective determination of sucrose based on electropolymerized molecularly imprinted polymer modified multiwall carbon nanotubes/glassy carbon electrode

A novel and selective electrochemical sensor was successfully developed for the determination of sucrose by integrating electropolymerization of molecularly imprinted polymer with multiwall carbon nanotubes. The sensor was prepared by electropolymerizing of o-phenylenediamine in the presence of template, sucrose, on a multiwall carbon nanotube-modified glassy carbon electrode. The sensor preparation conditions including sucrose concentration, the number of CV cycles in the electropolymerization step, pH of incubation solution, extraction time of template from the imprinted film and the incubation time were optimized using response surface methodology (RSM). A mixture of acetonitrile/acetic acid was used to remove the template. Hexacyanoferrate(II) was used as a probe to characterize the sensor using electrochemical impedance spectroscopy, cyclic voltammetry and differential pulse voltammetry. Capturing of sucrose by the modified electrode causes decreasing the response of the electrode to hexacyanoferrate(II). Calibration curve was obtained in the sucrose concentration range of 0.01–10.0mmolL−1 with a limit of detection 3μmolL−1. This sensor provides an efficient way for eliminating interferences from compounds with similar structures to sucrose. The sensor was successfully used to determine sucrose in sugar beet juices with satisfactory results.

Aptamer–protein binding detected by asymmetric flow field flow fractionation

Asymmetric flow field flow fractionation (AF4) should be suitable for the study of aptamer–target binding, because its gentle separation would impose little disturbance to the complex structure, and it can use carrier solutions with high salt concentrations to provide the most optimal interaction environment to the complex. However, no report has been found for such applications. Herein, we investigated the utility of AF4 as an effective tool for detection of the aptamer–protein complex. With the model system of human immunoglobulin E (IgE) and its aptamer, impacts on aptamer binding from the incubation and AF4 carrier solutions, as well as the flow conditions used during the sample focusing step, were studied. We found that the composition of the carrier solution, in particular, the presence of Mg2+, strongly influenced the complex's integrity in AF4. Also, the focusing conditions during sample injection in AF4 affected the binding equilibrium. Our findings highlight the necessity of maintaining the optimal binding environment during the time course of complex measurement; and demonstrated the good compatibility of AF4 with salty buffers and its high simplicity in conducting on-channel incubation. With its capability to carry out size-based separation of analytes with a wide range of dimensions, AF4 can be employed for detection of large proteins and even biological particles using aptamers. AF4 is also valuable for study of aptamer–target binding under different buffer environments for better understanding of the structure–function relationship of aptamers.

A systematic capillary electrophoresis study on the effect of the buffer composition on the reactivity of the anticancer drug cisplatin to the DNA model 2′-deoxyguanosine 5′-monophosphate (dGMP)

The development of DNA-targeted next-generation platinum-based anticancer chemotherapeutics is often accompanied by studies on the reactivity to DNA models. However, the incubation conditions used in literature vary widely, and some of the buffer/salts used are known to form complexes with Pt. Such coordination can influence the binding process and also the adducts formed. In a systematic approach, studies on the binding of cisplatin (1 mM) to dGMP (2 mM) in a series of different incubation solutions of relevance to biological systems are reported, employing capillary zone electrophoresis (CZE) with UV/vis and electrospray ionization-mass spectrometric (ESI-MS) detectors. Kinetic experiments performed with CZE-UV showed a high reactivity of dGMP to cisplatin in pure water (τ(1/2) = 4.1 ± 0.7 h) but a significantly slowed down in a solution containing a carbonate/phosphate buffer supplemented with NaCl, resulting in a half-life of dGMP of 25 ± 3 h. Especially carbonate had a major impact on the binding, though no coordination to the metal center was detectable with the methods used. The only adducts containing buffer components were (phosphate)Pt and tris(ammine)Pt species, as identified by means of CZE-ESI-MS, in addition to the main adduct [Pt(NH3)2(dGMP)2 - 4H(+)](2-) and other less abundant Pt-containing species.

Simultaneous determination of hair cortisol, cortisone and DHEAS with liquid chromatography–electrospray ionization-tandem mass spectrometry in negative mode

The present study aimed to develop a novel method for simultaneous assay of cortisol, cortisone and dehydroepiandrosterone sulfate (DHEAS) in human hair. The method was based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) in negative mode. Analytes were extracted from 20-mg hair incubated in 1ml of methanol for 5 days. 100μl non-SPE supernatant of the incubation solution was utilized in LC-MS/MS analysis. The liquid chromatography separation was performed on a reversed-phase C18 column with a mobile phase of 80:20 (v:v) methanol and deionized water containing 0.1% formic acid. The use of ESI in negative mode and the use of a small volume of the incubation solution significantly improved assay sensitivity. The linear range was 5-250pg/mg for cortisol and cortisone, and 5-500pg/mg for DHEAS. The limit of detection was 2pg/mg for the three analytes in hair. The coefficients of variation for intra-day and inter-day assay were less than 10%. The method was applied to determine the three analytes mentioned above of hair samples from 103 participants. The results indicated that there was no significant effect of age and education level on the hair cortisol, cortisone and DHEAS levels. The simple treatment procedure developed in the present study may facilitate simultaneous measurement of more steroids in hair.

A Multiwalled‐Carbon‐Nanotube‐Based Biosensor for Monitoring Microcystin‐LR in Sources of Drinking Water Supplies

AbstractA multiwalled carbon nanotube (MWCNT)‐based electrochemical biosensor is developed for monitoring microcystin‐LR (MC‐LR), a toxic cyanobacterial toxin, in sources of drinking water supplies. The biosensor electrodes are fabricated using vertically well‐aligned, dense, millimeter‐long MWCNT arrays with a narrow size distribution, grown on patterned Si substrates by water‐assisted chemical vapor deposition. High temperature thermal treatment (2500 °C) in an Ar atmosphere is used to enhance the crystallinity of the pristine materials, followed by electrochemical functionalization in alkaline solution to produce oxygen‐containing functional groups on the MWCNT surface, thus providing the anchoring sites for linking molecules that allow the immobilization of MC‐LR onto the MWCNT array electrodes. Addition of the monoclonal antibodies specific to MC‐LR in the incubation solutions offers the required sensor specificity for toxin detection. The performance of the MWCNT array biosensor is evaluated using micro‐Raman spectroscopy, including polarized Raman measurements, X‐ray photoelectron spectroscopy, cyclic voltammetry, optical microscopy, and Faradaic electrochemical impedance spectroscopy. A linear dependence of the electron‐transfer resistance on the MC‐LR concentration is observed in the range of 0.05 to 20 μg L−1, which enables cyanotoxin monitoring well below the World Health Organization (WHO) provisional concentration limit of 1 μg L−1 for MC‐LR in drinking water.

Preparation of Boronic Acid-Functionalized Mesoporous Nanomaterial and Its Application in Enrichment of Glycopeptides

A boronic acid-functionalized mesoporous nanomaterial was prepared by using a two-step post-grafting method. Firstly, the 3-glycidyloxypropyltrimethoxysilane (GLYMO) and 3-aminophenylboronic acid monohydrate (APB) were re- acted in oxyhydrogen sodium solution (pH 9.18) to prepare boronic-acid bonded GLYMO (denoted as GLYMO-APB). Sec- ondly, the MCM-41 was added into the prepared GLYMO-APB solution to prepare the final product of boronic acid func- tionalized MCM-41 (denoted as MCM-41-GLYMO-APB). This as-prepared material was characterized by FT-IR and the results showed that the boronic acid groups were successfully grafted to MCM-41. Glycopeptides can be enriched with high selectivity and high efficiency based on the formation of a cyclic diester between boronic hydroxyl and cis-diol on the glycan chain. The conditions of enriching glycopeptides in mixed digests of standard proteins by MCM-41-GLYMO-APB were compared and optimized. The incubation time, incubation solutions, washing ways and eluents used in the enrichment of glycopeptides were investigated. When using the ammonium bicarbonate buffer (100 mmol/L, pH 8.0) as the incubation so- lution and incubated for 1 h, the maximum number of enriched glycopeptides from horseradish peroxidase (HRP) digests could be obtained. After enrichment, washing the prepared material with the incubation solution twice for 5 min, nonglyco- peptides interference can be in a large extent removed. The enriched glycopeptides binding to the surface of MCM-41-GLYMO-APB could be released when using 1% trifluoroacetic acid containing 2,5-dihydroxybenzoic acid as the eluent, the operating steps could be simplified when using this optimized eluent because there was no need of adding the MALDI matrix solution afterwards. Therefore, this optimized protocol provided a reference for the glycopeptide enrichment with boronic acid-functionalized materials, and it also laid the foundation for the research of utilizing boronic acid-based chemical method to the glycopeptide enrichment in biological samples. Meanwhile, the highly ordered hexagonal cylindrical mesoporous structure of MCM-41-GLYMO-APB provided the possibility of being used for the research of glycopeptidome in biological samples. Keywords glycoproteins; boronic acid-functionalized mesoporous nanomaterial; enrichment; mass spectrometry

Effects of 5-aminolevulinic acid-mediated sonodynamic therapy on macrophages

BackgroundInflammatory cells exhibit an elevated level of protoporphyrin IX (PpIX) after the administration of 5-aminolevulinic acid (ALA). Here, we investigate the sonodynamic effects of ALA-derived PpIX (ALA-PpIX) on macrophages, which are the pivotal inflammatory cells in atherosclerosis.Methods and resultsCultured THP-1 macrophages were incubated with ALA. Fluorescence microscope and fluorescence spectrometer detection showed that intracellular PpIX increased with the concentration of ALA in the incubation solution in a time dependent manner; the highest level of intracellular PpIX was observed after 3-hour incubation. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays demonstrated that lower concentrations (less than 2 mM) of ALA have no influence on cell viability (more than 90% of cells survived), but sonodynamic therapy (SDT) with a low concentration of ALA significantly decreased the survival rate of cells, and the effect was increased with both ALA concentration and ultrasound exposure time. Cell apoptosis and necrosis induced by ALA-mediated SDT (ALA-SDT) were measured using Hoechst 33258 and propidium iodide assay. ALA-SDT induced both cell apoptosis and necrosis, and the maximum apoptosis/necrosis ratio was observed at 6 hours after SDT with 1 mM of ALA and 5 minutes of ultrasound exposure. Flow cytometry analysis showed that ALA-SDT significantly increased late stage apoptotic cells (about 10-fold control). Furthermore, ALA-SDT induced reactive oxygen species generation in THP-1 macrophages immediately after the treatment and a conspicuous loss of mitochondrial membrane potential (MMP) at 6 hours compared with that of the control, ALA alone, and ultrasound alone groups.ConclusionALA-SDT exhibited synergistic apoptotic effects on THP-1 macrophages, involving excessive intracellular reactive oxygen species generation and MMP loss. Therefore, ALA-SDT is a potential treatment for atherosclerosis.

Application of Carbon Quantum Dots in Fluorescent Imaging of Human Serum Proteins after Polyacrylamide Gel Electrophoresis

A new method of carbon quantum dots (CQDs) fluorescent imaging for human serum proteins detection after polyacrylamide gels electrophoresis (PAGE) is established. Polyacrylamide gel electrophoresis is one of the most general and powerful technique to separate complex biosamples, and it is widely used in molecular biology, biochemistry and medicine. With the evolution of clinical proteomics, the development of a novel method to detect serum proteins after PAGE with high resolution and high sensitivity is of great significant. As far as we know, the carbon quantum dots have not been applied in the detection of serum proteins after PAGE. The fluorescent carbon quantum dots were synthesized by a one-step microwave pyrolysis method: glycerol and phosphate buffer (7.1 mmolL -1 , pH 7.4) (φ=70%) was mixed evenly, and then put the solu- tion into the advanced microwave digestion system and heated for 14 min (750 W), the colorless solution turned to yellow after reaction. The yellow solution was diluted into the incubation solution with HOAc-NaOAc buffer (80 mmolL -1 , pH 2.7), and then used for staining serum proteins. The emission and excitation spectra of carbon quantum dots were measured, and the excitation wavelength allowed the use of an ultraviolet lamp at 365 nm for the fluorescent imaging. In order to inves- tigate the performance of CQDs fluorescent imaging, the dilution of human serum samples separated by electrophoresis was used for detection, and was compared with traditional method of CBB-R250 staining and silver staining. The results sug- gested that CQDs based on fluorescent imaging could effectively detect human serum proteins with high sensitivity and resolution. The sensitivity of CQDs imaging was higher than CBB staining and comparable to silver staining. Therefore, the CQDs fluorescent imaging could be an inexpensive, time-saving, pollution-free and convenient method with high sensitivity and resolution for the detection of human serum proteins. It demonstrates the CQDs fluorescent imaging technology has great significant prospects in the development of biotechnology and nanotechnology. Keywords carbon quantum dot; polyacrylamide gel electrophoresis; protein; fluorescent imaging

Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry for Identification of In Vitro and In Vivo Metabolites of Bornyl Gallate in Rats

Bornyl gallate (BG) is a potential drug candidate synthesized by the reaction of two natural products, gallic acid and borneol. Previous studies have strongly suggested that BG is worthy of further investigation due to antioxidant, antiatherosclerosis activities, and obvious activity of stimulating intersegmental vessel growth in zebrafish. This work was designed to elucidate the metabolic profile of BG through analyzing its metabolites in vitro and in vivo by a chromatographic separation coupled with a mass spectrometry. The metabolites of BG were characterized from the rat liver microsome incubation solution, as well as rat urine and plasma after oral administration. Chromatographic separation was performed on an Agilent TC-C18 column (250 mm × 4.6 mm, 5 μm) with gradient elution using methanol and water containing 0.2% (V : V) formic acid as the mobile phase. Metabolites identification involved analyzing the retention behaviors, changes of molecular weights and MS/MS fragment patterns of BG and the metabolites. Five compounds were identified as isomers of hydroxylated BG metabolites in vitro. The major metabolites of BG in rat urine and plasma proved to be BG-O-glucuronide and O-methyl BG-O-glucuronide. The proposed method confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of BG.

Laboratory research on the contaminants release during the anaerobic decomposition of high-density cyanobacteria

The anaerobic decomposition of high-density cyanobacteria and the kinetics of contaminants release were investigated based on a set of batch tests in this study.The results showed that the degradation rates of chlorophyll-a(Chl.a were 0.074,0.133,and 0.081 d-1,respectively,at different cyanobacterial densities of 2.23×1012,1.19×1013 and 4.47×1013 cells/L,respectively.The chemical oxygen demand increased while the UV254 value increased initially and then decreased,suggesting that the release of organic compounds increased,but the molecules of which were reduced gradually during the degraded process.Accordingly,the pH of incubation solution remained acidic and the conductance increased,up to 949 μS/cm.The decomposition of cyanobacteria resulted in a high release of nitrogen and phosphorus.Moreover,dissolved organic nitrogen was degraded gradually into inorganic nitrogen form,in which the amount of ammonia nitrogen accounts for 90%.The results demonstrate that the anaerobic decomposition of high-density cyanobacteria can release a large amount of organic compounds and nutrients into water,furthermore the release of contaminants increases with the increasing density of cyanobacteria.Therefore,to avoid high-density cyanobacteria deteriorating water quality and even threatening drinking water safety,we need to remove cyanobacteria timely when algal bloom occurs in the lake which is served as water resource.

Participation of extracellular calcium in α-hederin-induced contractions of rat isolated stomach strips

Ethnopharmacological relevanceThe dry extract of Hedera helix leaves, due to its secretolytic and antispasmodic effects, is commonly used to produce pharmaceuticals applied in case of cough and other respiratory symptoms. The results of some in vitro studies as well as the clinical signs of poisoning caused by Hedera helix suggest however strong contractile effect on smooth muscle. In order to clarify the impact of α-hederin (the main active agent of ivy extract) on smooth muscle, the origin of activated calcium involved in α-hederin-induced contraction of gastric smooth muscle preparations was studied. Materials and methodsThe study was carried out on rat isolated stomach corpus and fundus strips, under isotonic conditions. The effect of α-hederin (100μM) on smooth muscle preparations was measured before and after the treatment with verapamil during the incubation in modified Krebs–Henseleit solution (M K–HS). Besides, the effect of saponin was measured during the incubation of preparation in Ca2+-free modified Krebs–Henseleit solution or Ca2+-free EGTA-containing modified Krebs–Henseleit solution. ResultsThe obtained results revealed that the application of verapamil significantly inhibited the reaction evoked by α-hederin. The incubation of stomach strips in calcium-free modified Krebs–Henseleit solution did not change the force of the observed contraction in comparison to the reaction of the preparations incubated in regular incubation solution (M K–HS). In contrary, the replacement of M K–HS by calcium-free chelator-containing solution inhibited totally the reaction to α-hederin. ConclusionsThe results indicated that α-hederin-induced contraction results from the influx of calcium which is located in intercellular spaces or bound to the outside of the cell membrane. The Ca2+ influx occurs predominantly through voltage-dependent calcium channels of L-type.

Physiological engineering of Pseudomonas aurantiaca antimicrobial activity: effects of sodium chloride treatment

The influence of sodium chloride (NaCl) treatment on the antifungal activity of the bacterium Pseudomonas aurantiaca, a producer of biopesticide for vegetable plants, was investigated. It was shown that an increase in the NaCl concentration in incubation solutions from 1 M to 3 M led to a significant increase in the antifungal activity of this bacterium. Antifungal activity continued to increase with prolonged treatment of bacteria in fresh nutrient medium from 72 h to 96 h. These findings could be very important for the further development of biotechnological processes directed not only to the production of new active biopesticides but also of other valuable resources.

Femtomolar detection of single mismatches by discriminant analysis of DNA hybridization events using gold nanoparticles

Even though DNA-based nanosensors have been demonstrated for quantitative detection of analytes and diseases, hybridization events have never been numerically investigated for further understanding of DNA mediated interactions. Here, we developed a nanoscale platform with well-designed capture and detection gold nanoprobes to precisely evaluate the hybridization events. The capture gold nanoprobes were mono-laid on glass and the detection probes were fabricated via a novel competitive conjugation method. The two kinds of probes combined in a suitable orientation following the hybridization with the target. We found that hybridization efficiency was markedly dependent on electrostatic interactions between DNA strands, which can be tailored by adjusting the salt concentration of the incubation solution. Due to the much lower stability of the double helix formed by mismatches, the hybridization efficiencies of single mismatched (MMT) and perfectly matched DNA (PMT) were different. Therefore, we obtained an optimized salt concentration that allowed for discrimination of MMT from PMT without stringent control of temperature or pH. The results indicated this to be an ultrasensitive and precise nanosensor for the diagnosis of genetic diseases.

Degradation Kinetics of an Aspartyl-Tripeptide-Derived Diketopiperazine under Forced Conditions

The aim of the present study was the determination of the stability of a diketopiperazine (DKP) derived from the aspartyl tripeptide Phe-Asp-GlyOH at pH 10 and 80°C and 25°C as well as at pH 7.4 and 80°C. The analysis was performed using a validated capillary electrophoresis assay. Rapid epimerization of the incubated cis-DKP to the trans-DKP was observed under all conditions. Linear diastereomeric α-l/d-Asp and β-l/d-Asp peptides were the primary reaction products at pH 10 and 80°C, indicating that a cyclic succinimide is formed in the process. In contrast, the DKPs were by far the major compounds in the incubation solutions at pH 10 and 25°C and at pH 7.4 and 80°C, whereas the linear Asp peptides were found only at low concentrations. A kinetic model was derived to fit the concentration versus time data, which consider the succinimide as central intermediate for DKP formation and for isomerization and enantiomerization of the linear Asp peptides. Besides the back reaction of the DKPs to the succinimides, an additional hydrolysis reaction of the DKP ring was considered to obtain the fit of the experimental data, indicating that additional degradation reactions have to be considered for Asp-derived DKPs.

Efficient degradation of methyl orange via multilayer films of titanium dioxide and silicotungstic acid

We report layer-by-layer (LbL) assembly of TiO2 and H4SiW12O40 (SiW12) multilayer film on silicon wafers and glass slides for photocatalytic degradation of methyl orange (MO). The photocatalytic efficiency of the obtained multilayer film increases along with the decrease of pH and salt concentration of the incubation solution. The results show that MO can be almost removed in pH 2.0 solution without salt addition in the first 60 min incubation when MO concentration is lower than 15 mg/L. Different salts show an apparent inhibitory effect on photocatalytic degradation of MO with the order of ZnCl2>KCl>NaCl>LiCl. The TiO2/SiW12 multilayer film maintains photocatalytic activity even after five degradation cycles. The reaction of MO photodegradation accords with an apparent first-order dynamics.

The dynamic nature of incubation solution after cooling to room temperature in amyloid formation of hen egg white lysozyme: An FTIR assessment

Amyloid formation of hen egg white lysozyme (HEWL) usually requires elevated temperature, while biophysical characterizations on the incubation solution are often performed at room temperature. Whether maintaining the incubation solution at room temperature results in further structural changes is a significantly important issue that has never been explored. Herein, we use FTIR spectroscopy to assess this issue and reveal that the hot incubation solution of HEWL after cooling to room temperature is in a dynamically evolving state and forms β-sheet aggregates continuously over time. Combined with AFM, we show that these aggregates are non-fibrillar β-sheet aggregates and have vibrational signature distinct from that of fibrillar aggregates. Using FTIR difference spectroscopy, we demonstrate that these non-fibrillar aggregates are in an anti-parallel β-sheet configuration. We also provide a detailed discussion on the spectral assignments for protein aggregates in anti-parallel and parallel β-sheet configurations. With FTIR second derivative technique, we show that these non-fibrillar aggregates are in fact present along with fibrillar aggregates during incubation under elevated temperature but are less stable compared with that at room temperature. Implications from the current work are discussed.