L-cysteine Self-assembled Monolayers Research Articles

The Effect of Counterions on the Detection of Cu2+ Ions in Aqueous Solutions Using Quartz Tuning Fork (QTF) Sensors Modified with L-Cysteine Self-Assembled Monolayers: Experimental and Quantum Chemical DFT Study

In this study, a sensing device employing a gold-coated quartz tuning fork (QTF) modified with a self-assembled monolayer (SAM) of L-cysteine was evaluated for the sensitive detection of Cu2+ ions in aqueous solutions. Three copper (II) salts, CuSO4, CuCl2, and Cu(NO3)2, at four different concentrations (10−12, 10−10, 10−8, and 10−6 M) in small (100 μL) water sample amounts were each used as analytes to investigate the influence of their counterions in the detection of the Cu2+ ions. It was found that, among the counterions, the sulfate anion had the largest effect upon the detection of Cu2+ in water, in the following order: SO42− > Cl− > NO3−. The lower limit of detection of the Cu2+ ions detected was in the 10−12 M range. The frequency shifts measured with the QTFs relative to deionized water were inversely proportional to the concentration/mass of the analytes. Density functional theory calculations were conducted to understand the effect of the counterions on the respective electronic interaction energies for the apparent host–guest binding of the analytes with L-cysteine and with gold surface-bound L-cysteine molecules. Gas phase (both with and uncorrected BSSE) and solution phase interaction energies (ΔIE) calculated at the B3LYP/LANL2DZ and ωB97XD levels of theory showed that the stability for the complexes were in the following order: [L-cysteine]⊃[CuSO4] > [L-cysteine]⊃[CuCl2] > [L-cysteine]⊃[Cu(NO3)2], which supports our experimental findings, as they were in the same order as the experimentally observed order for the copper salts tested: CuSO4 > CuCl2 > Cu(NO3)2.

An origami paper-based electrochemical immunoassay for the C-reactive protein using a screen-printed carbon electrode modified with graphene and gold nanoparticles

An origami paper-based electrochemical immunoassay for C-reactive protein (CRP) detection is described. The assay integrates multiple steps of electrode modification into a single device. A graphene-modified screen-printed carbon electrode (G/SPCE) was employed to enhance sensitivity. Gold nanoparticles were first electrodeposited onto the G/SPCE, followed by a self-assembled monolayer of L-cysteine. The capture anti-CRP was then covalently immobilized on the modified electrode. CRP was quantified by measuring the changes in the charge-transfer resistance of the electrode by using hexacyanoferrate as the redox probe. Cyclic voltammetry and scanning electron microscopy were also applied to verify the successful modification of the electrode. Under optimal conditions, impedance increase in the 0.05-100μgmL-1 CRP concentration range, and the limit of detection is 15ngmL-1 (at S/N = 3). The immunoassay was successfully applied to the determination of CRP in a certified human serum sample. This method is simple, low-cost, portable and disposable. Graphical abstract An origami paper-based analytical device (oPAD) is described that integrates the multistep of electrode modification, immobilization and detection into a single device. The direct conjugation between the capture antibody and target molecule was allowed to use in this system. The C-reactive protein (CRP) concentration in serum samples was determined using electrochemical impedance spectroscopy.

Voltammetric Biosensor Based on Nitrogen-doped Ordered Mesoporous Carbon for Detection of Organophosphorus Pesticides in Vegetables

Background: Pesticides residues in agricultural products have posed a serious threat to food safety and human health, so it is necessary to develop a rapid and accurate method to detect pesticide in the environment. N-OMC with excellent electroconductivity, high biocompatibility and the functional amino group that can be covalently attached to the enzyme can be applied to construct a sensitive and stable acetylcholinesterase biosensor for rapid and accurate detection of organophosphorus pesticides with the help of L-cysteine self-assembled monolayer and AuNPs. Methods: Transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy and nitrogen adsorption measurements are used to characterize materials. Electrochemical impedance spectroscopy and cyclic voltammetry are used to study the surface features of modified electrodes. Differential pulse voltammetric is used to measure the peak current of modified electrodes. GC-MS is applied to verify the reliability of the prepared biosensor for organophosphorus pesticides detection. Results: N-OMC was synthesized and applied to constructed stable and sensitive acetylcholinesterase biosensors. The combination of N-OMC, L-cysteine self-assembled monolayer and AuNPs to modify the electrode surface has greatly improved the conductivity of biosensor and provided a stable platform for acetylcholinesterase immobilization. The linear detection range of paraoxon was from 3 to 24 nM with a lower detection limit of 0.02 nM. Conclusion: The biosensor exhibited satisfactory reproducibility, repeatability and stability, and was successfully employed to determine the paraoxon in vegetables as well as tap water samples, providing a promising tool for rapid and sensitive detection of organophosphorus pesticides in agricultural products.

Chitosan-Based Quartz Crystal Microbalance for Alcohol Sensing

Short-chain alcohols are a group of volatile organic compounds (VOCs) that are often found in workplaces and laboratories, as well as medical, pharmaceutical, and food industries. Real-time monitoring of alcohol vapors is essential because exposure to alcohol vapors with concentrations of 0.15–0.30 mg·L−1 may be harmful to human health. This study aims to improve the detection capabilities of quartz crystal microbalance (QCM)-based sensors for the analysis of alcohol vapors. The active layer of chitosan was immobilized onto the QCM substrate through a self-assembled monolayer of L-cysteine using glutaraldehyde as a cross-linking agent. Before alcohol analysis, the QCM sensing chip was exposed to humidity because water vapor significantly interferes with QCM gas sensing. The prepared QCM sensor chip was tested for the detection of four different alcohols: n-propanol, ethanol, isoamyl alcohol, and n-amyl alcohol. For comparison, a non-alcohol of acetone was also tested. The prepared QCM sensing chip is selective to alcohols because of hydrogen bond formation between the hydroxyl groups of chitosan and the analyte. The highest response was achieved when the QCM sensing chip was exposed to n-amyl alcohol vapor, with a sensitivity of about 4.4 Hz·mg−1·L. Generally, the sensitivity of the QCM sensing chip is dependent on the molecular weight of alcohol. Moreover, the developed QCM sensing chips are stable after 10 days of repeated measurements, with a rapid response time of only 26 s. The QCM sensing chip provides an alternative method to established analytical methods such as gas chromatography for the detection of short-chain alcohol vapors.

Ultrasensitive Detection of Cu2+ Using a Microcantilever Sensor Modified with L-Cysteine Self-Assembled Monolayer.

A microcantilever was modified with a self-assembled monolayer (SAM) of L-cysteine for the sensitively and selectively response to Cu(II) ions in aqueous solution. The microcantilever undergoes bending due to sorption of Cu(II) ions. The interaction of Cu(II) ions with the L-cysteine on the cantilever is diffusion controlled and does not follow a simple Langmuir adsorption model. A concentration of 10-10M Cu(II) was detected in a fluid cell using this technology. Other cations, such as Ni2+, Zn2+, Pb2+, Cd2+, Ca2+, K+, and Na+, did not respond with a significant deflection, indicating that this L-cysteine-modified cantilever responded selectively and sensitively to Cu(II).

Electrochemical Manipulation of Living Cells Supported By Polymeric Nanosheets

Introduction Regenerative medicine based on cell sheets has been widely investigated. Developing an effective and minimally invasive cell transplantation process is an important enabling step for such medical treatments. However, cell sheets are fragile and handling them is difficult. Recently, we have investigated the use of biodegradable polymeric nanosheets for introducing cells into the body and found that the flexible nanosheets on which cells are cultured can be aspirated and injected by a syringe needle without significant loss of cell viability. To harvest cell/nanosheet constructs from a substrate, a water soluble sacrificial layer such as polyvinyl alcohol (PVA) was used, however, a drawback in this method is that the timing to detach the cell/nanosheet constructs from the substrate cannot be controlled. In this paper, we use an electrochemical method for harvesting the cell/nanosheet constructs in vitro. Cells are cultured on nanosheets attached on a self-assembled monolayer (SAM) of alkanethiol. The SAM is reductively desorbed from the gold substrate by the application of a negative electrical potential, which detaches the cell/nanosheet constructs from the gold surface in a rapid manner. The harvested cell/nanosheet constructs were transplanted into the subretinal space of rat eyes through syringe needles. Material and Method A gold layer was patterned by sputter-depositing gold with a patterned silicon mask. The gold surface was modified with 10-carboxy-1-decanethiol, 7-carboxy-1-heptanethiol or L-cysteine by immersing the substrates in any of the three thiol solutions for 30 min at room temperature. The desorption of the SAMs was performed and analyzed electrochemically. Nanosheets were first prepared on PVA-coated substrates by a combination of a spincoating and microcontact printing technique using poly(dimethyl siloxane) (PDMS) stamps. A solution of poly(lactic-co-glycolic acid) (PLGA) was spincoated onto the PDMS stamp and the resulting PLGA layer was transferred onto a PVA-coated substrate. Freestanding nanosheets were obtained by dissolving the PVA sacrificial layer with water and attached on the SAM-modified gold surface. To desorb the nanosheets with SAMs, a voltage of 1.5 V, supplied by an AA-size dry battery, was applied between the gold electrode (cathode) and the Pt wire (anode) in phosphate buffered saline. Retinal pigment epithelium (RPE) cells were cultured on PLGA nanosheets attached on the SAMs for 2 days to form cell/nanosheet constructs. Electrochemical desorption of SAMs with cell/nanosheet constructs was conducted in culture medium. Sprague-Dawley rats (SLC) weighing 250-300 g were used in this study. After the rats were anesthetized with ketamine hydrochloride and xylazine hydrochloride, RPE cell/nanosheet constructs were injected into the subretinal space using a hand-made glass capillary needle. Result and Discussion Cyclic voltammograms observed in the reductive desorption of SAMs showed a clear dependence of peak potential on the length of the alkyl chain. With longer chains, the peak potential shifted to the negative direction, which has been explained by van der Waals interactions between the alkyl chains. Since the SAM of L-cysteine was reductively desorbed at a lower potential (around -0.7 V vs. Ag/AgCl) than the SAM of the two other thiol molecules and L-cysteine is biocompatible, the SAM of L-cysteine was used for harvesting nanosheets and cell/nanosheet constructs. PLGA nanosheets attached on the SAM of L-cysteine were successively detached from the substrate by gently pipetting within 1 min after applying the reductive potential to the gold electrode. On the other hand, nanosheets on the SAM without applying the voltage did not detach from the substrate. In a similar manner, RPE cell/nanosheet constructs could be detached from the substrate and the live/dead staining showed the cells on the nanosheets are viable. Finally, we used our system to deliver RPE cell/nanosheet constructs to the subretinal space of a rat eye in vivo. Nanosheets were injected with 2 μL of saline to the subretinal space through the sclera using a glass capillary needle. Optical coherence tomography (OCT) images of the retina taken one week after the injection of the nanosheets showed a shadow of the sheet like structure in the subrentinal space while there was not such shadow in the OCT image of a control. Also, a shadow of the circular nanosheet was observed at the posterior segment of the isolated eye. These results indicate that the nanosheets were successfully injected and spread into the subretinal space of rat eyes. Conclusion We reported a method for noninvasively harvesting cell/nanosheet constructs from a culture surface by using the electrochemical desorption of SAM. The harvested RPE/nanosheet constructs could be delivered to the subretinal space using a capillary needle. The harvesting and delivering system of cell/nanosheets constructs could be a useful tool for cell transplantation.

A highly sensitive electrochemical determination of norepinephrine using l-cysteine self-assembled monolayers over gold nanoparticles/multi-walled carbon nanotubes electrode in the presence of sodium dodecyl sulfate

A novel electrochemical sensor consisting of l-cysteine self-assembled monolayers over gold nanoparticles/multi-walled carbon nanotubes modified glassy carbon electrode (l-Cys/AuNPs/MWCNTs/GCE) was fabricated and applied for the determination of norepinephrine (NE) in the presence of sodium dodecyl sulfate (SDS) for the first time. Electroanalytical measurements including cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used. The optimum buffer of pH 5.0 contained 30μM SDS, the NE oxidation peak current enhanced linearly with concentration ranging from 0.2 to 100μM (R2 of 0.9998) with a detection limit of 0.03μM. In the presence of SDS, the modified l-Cys/AuNPs/MWCNTs/GCE electrode showed high selectivity for the detection of NE in the presence of ascorbic acid and uric acid. High recoveries in the range of 80.7–100.2% were obtained in spiked human serum samples. Our modified electrode displayed a high reproducibility, sensitivity and long term stability.

Low-energy electron-induced dissociation in condensed-phase L-cysteine I: Desorption of anions from chemisorbed films

Among amino acids, cysteine has been widely studied, becoming a standard for molecular self-assembly experiments, because its mercapto group (-SH) allows the formation of self-assembled monolayers (SAMs) on metal surfaces. Dissociative electron attachment (DEA) on L-cysteine SAMs is investigated utilizing a time-of-flight mass spectrometer coupled with a low-energy electron gun. The results show that electrons with kinetic energies of 3 to 15 eV attach to L-cysteine producing anionic fragments of different masses (e.g., H-, O-, OH-, S-, SH-) via dissociation of intermediate transient anions. The anion yield functions exhibited purely resonant behaviour with electron energies below 15 eV, indicating that the formation of transient anions is the predominant mechanism of production of anionic fragments from L-cysteine dissociation.

Detection of prostate specific antigen based on electrocatalytic platinum nanoparticles conjugated to a recombinant scFv antibody

Highly sensitive and label free detection of prostate specific antigen (PSA) still remains a challenge in prostate cancer diagnosis. In this paper, we propose a sensitive electrochemical immunosensor based on electrocatalytic platinum nanoparticles conjugated to a recombinant scFv antibody. Gold disc electrodes functionalised with a l-Cysteine (Cys) self-assembled monolayer (SAM) were used to covalently bind PSA specific monoclonal antibody (anti-PSA) using N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. Immunosensing was completed using sandwich-type immunoreaction of the PSA–antigen (1–30ng/mL) between anti-PSA immobilized on the l-Cys modified electrode using label free electrochemical impedance (EIS) technique. Furthermore, highly specific in-house generated scFv fragments as receptor proteins were utilised for one step site-directed immobilisation on the surface of platinum nanoparticles (PtNPs). To improve the sensitivity of the immunoassay, these scFV labelled electrocatalytic PtNPs were then used for covalent hybridisation to the PSA modified electrode and then applied in a hybridisation assay to determine the concentration of the PSA by measuring the faradaic current associated with reduction of peroxide in solution. Semi-log plots of the PSA concentration vs. faradaic current are linear from 1 to 30ng/mL and pM concentrations can be detected without the need for molecular, e.g., PCR or NASBA, amplification.

L-cysteine/polydopamine nanoparticle-coatings for copper corrosion protection

Polydopamine (PDA) coating on a copper surface was successfully prepared using a two-step process. In the first stage, in order to improve the adhesion of PDA on the copper surface, a self-assembled monolayer of L-cysteine was deposited on a bare copper. Thereafter, the PDA coating was fabricated through the chemical adsorption and auto-polymerization of the dopamine hydrochloride on the L-cysteine-coated copper surface. The inhibitory behavior of L-cysteine and PDA films has been investigated in aqueous 3.5% NaCl solution using potentiodynamic polarization and electrochemical impedance spectroscopy. It is demonstrated that the L-cysteine/PDA coating provides significant protection against copper corrosion.

A biomimetic platform to study the interactions of bioelectroactive molecules with lipid nanodomains.

In this work, we developed a biomimetic platform where the study of membrane associated redox processes and high-resolution imaging of lipid nanodomains can be both performed, based on a new functional gold modification, l-cysteine self-assembled monolayer. This monolayer proved to be ideal for the preparation of defect-free planar supported lipid bilayers (SLBs) where nanodomains with height difference of ∼1.5 nm are clearly resolved by atomic force microscopy. Single and multicomponent lipid compositions were used, leading to the formation of different phases and domains mimicking the lateral organization of cellular membranes, and in all cases stable and continuous bilayers were obtained. These platforms were tested toward the interaction with bioelectroactive molecules, the antioxidant quercetin, and the hormone epinephrine. Despite the weak interaction detected between epinephrine and lipid bilayers, our biomimetic interface was able to sense the redox process of membrane-bound epinephrine, obtain its surface concentration (9.36 × 10(-11) mol/cm(2) for a fluid bilayer), and estimate a mole fraction membrane/water partition coefficient (Kp) from cyclic voltammetric measurements (1.13 × 10(4) for a fluid phase membrane). This Kp could be used to quantitatively describe the minute changes observed in the photophysical properties of epinephrine intrinsic fluorescence upon its interaction with liposome suspensions. Moreover, we showed that the lipid membrane stabilizes epinephrine structure, preventing its oxidation, which occurs in neutral aqueous solution, and that epinephrine partition and mobility in membranes depends on lipid phase, expanding our knowledge on hormone membrane interactions.

Direct electrochemistry and intramolecular electron transfer of ascorbate oxidase confined on l-cysteine self-assembled gold electrode

A direct electrochemistry and intramolecular electron transfer of multicopper oxidases are of a great importance for the fabrication of these enzyme-based bioelectrochemical-devices. Ascorbate oxidase from Acremonium sp. (ASOM) has been successfully immobilized via a chemisorptive interaction on the l-cysteine self-assembled monolayer modified gold electrode (cys-SAM/AuE). Thermodynamics and kinetics of adsorption of ASOM on the cys-SAM/AuE were studied using cyclic voltammetry.A well-defined redox wave centered at 166±3mV (vs. Ag│AgCl│KCl(sat.)) was observed in 5.0mM phosphate buffer solution (pH7.0) at the fabricated ASOM electrode, abbreviated as ASOM/cys-SAM/AuE, confirming a direct electrochemistry, i.e., a direct electron transfer (DET) between ASOM and cys-SAM/AuE. The direct electrochemistry of ASOM was further confirmed by taking into account the chemical oxidation of ascorbic acid (AA) by O2 via an intramolecular electron transfer in the ASOM as well as the electrocatalytic oxidation of AA at the ASOM/cys-SAM/AuE.Thermodynamics and kinetics of the adsorption of ASOM on the cys-SAM/AuE have been elaborated along with its direct electron transfer at the modified electrodes on the basis of its intramolecular electron transfer and electrocatalytic activity towards ascorbic acid oxidation and O2 reduction. ASOM saturated surface area was obtained as 2.41×10−11molcm−2 with the apparent adsorption coefficient of 1.63×106Lmol−1. The ASOM confined on the cys-SAM/AuE possesses its essential enzymatic function.

Synthesis of Nanoporous Gold Electrode and Its Application in Electrochemical Sensor

Abstract In ammonia solution, binary gold-silver alloy films were directly deposited onto indium tin oxide (ITO) substrate surface by using electrochemical method with chloroauric acid and silver nitrate as precursors. Then high-surface-area nanoporous gold (NPG) film modified electrodes were fabricated by chemical dealloying of the less noble component silver from the surface. The modified electrodes were characterized. The electrode prepared can be successfully functionalized with self-assembled monolayers of L-cysteine, which can be applied to the sensitive and selective determination of Cu(II). Under the optimal conditions and 5 min of absorption time for Cu(II) ions, the cathodic current is linearly proportional to Cu(II) concentration ranged from 0.05 to 4 μM with a detection limit of 0.03 μM. The relative standard deviation is 4.3% for 1 μM Cu(II) solution. The established method was successfully applied for the determination of Cu(II) in water samples of environment.

Electrochemical determination of trace copper(II) with enhanced sensitivity and selectivity by gold nanoparticle/single-wall carbon nanotube hybrids containing three-dimensional l-cysteine molecular adapters

This paper described how a inorganic–organic hybrids electrochemical sensor, a three-dimensional l-cysteine self-assembled monolayers (SAM) functionalized gold nanoparticles/single-walled carbon nanotubes/glassy carbon electrode (l-cys/AuNPs/SWCNTs/GCE), was applied to improve the performance of detecting trace Cu(II) in stripping voltammetry. The AuNPs/SWCNTs nanohybrid has a three-dimensional porous nanostructure with large active surface area, and the l-cysteine adapters on AuNPs/SWCNTs surface can greatly enhance the sensitivity and selectivity in detecting Cu(II). The differential pulse anodic stripping voltammetry (DPASV) response of the Cu(II) at the l-cys/AuNPs/SWCNTs/GCE was ca. 6.7 and 6.5 times larger than that at the AuNPs/SWCNTs/GCE and l-cys/AuNPs/GCE without SWCNTs, respectively. The sensor demonstrated a wide linear response range and a lower detection limit of 0.02nM with a signal-to-noise of 3 using 3min of preconcentration time. The interference experiments showed that Ag(I), Pb(II), Cd(II) and Hg(II) had little influence on Cu(II) signal. The high sensitivity and excellent selectivity in contrast to the values reported previously in the area of electrochemical Cu(II) detection, demonstrated the analytical performance of the as-prepared sensor toward Cu(II) was superior to the existing electrodes. The as-prepared sensor was further applied to determine the Cu(II) in the real environmental water sample, and the results agreed satisfactorily with the certified values.

Synthesis of Nanoporous Gold Electrode and Its Application in Electrochemical Sensor

In ammonia solution,binary gold-silver alloy films were directly deposited onto indium tin oxide(ITO) substrate surfaces using electrochemical method from chloroauric acid and silver nitrate.Then high-surface-area nanoporous gold(NPG) film modified electrodes were fabricated by chemical dealloying of the less noble component silver from the surface.The modified electrodes were characterized.The electrode can be successfully functionalized with self-assembled monolayers of L-cysteine,which can be applied to the sensitive and selective determination of Cu2+.Under the optimal conditions and 5 min of absorption time for Cu2+ ions,the cathodic current was linear proportional with Cu2+ concentration from 0.05 to 4 μmol/L with a detection limit of 0.03 μmol/L.The relative standard deviation was 4.3% for 1 μmol/L Cu2+ solution.The established method was successfully applied for the determination of Cu2+ in water sample of environment.

Effect of Boric Acid on the Crystallization of L-Lysine Monohydrochloride Dihydrate on Self-Assembled Monolayers of L-Cysteine

Effect of Boric Acid on the Crystallization of L-Lysine Monohydrochloride Dihydrate on Self-Assembled Monolayers of L-Cysteine

Potential-Induced Conformational Changes in Self-Assembled Monolayers of L-Cysteine at p-GaAs(100) Electrodes

not Available.

Electrochemical investigation on the film of L-cysteine self-assembled to nanoparticles on a gold electrode

The film contained L-cysteine and gold nanoparticles were provided by self-assembled monolayers (SAMs) and potentiostatic electrodeposition technology on the gold electrode. Two methods were used to study the film: In the first, cyclic voltammetry (CV) was used to inspect the functional groups of the film and the same time hydroquinone was chosen to be a probe molecule in the based solution; secondly, based on analytical technology of scanning electrochemical microscopy (SECM), the heterogeneous rate constant (keff) between solid phase (the modified electrode) and liquid phase (K3Fe(CN)6) was obtained. As a result, the better binary catalysis of hydroquinone was demonstrated and the heterogeneous rate constant (keff) is the greater at 8 h for L-cysteine self-assembled monolayers (SAMs).

Calixarene Molecules Immobilized on Gold Substrates of QCM Sensors Based on Self-Assembled Monolayer Technology

Four calixarene supramolecular compounds, RCT, PCT, MRCT, and TBCA as active materials, were immobilized on gold substrates of quartz crystal microbalance (QCM) sensors by using L-cysteine self-assembled monolayer (SAM) as a linker. The RCT immobilized QCM sensor possessed the best response characteristics for methanol molecule when the assembling concentration of RCT reached 1.0 mg/mL. The frequency shift response value of the RCT immobilized QCM sensor was in direct proportion to the concentration of methanol vapor with a range of 0 ~ 6000 ppm. Comparing with a gas chromatography, the proposed QCM sensor could be well used for the determination of methanol vapor with a recovery rate of 98.01 ~ 103.9 %, and the two methods showed a well consistent examination result. Also, the sensor possessed good reproducibility and stability, showing that the RCT immobilized QCM sensor can be applied for the detection of the methanol vapor of atmospheric polutants in our living environment.

Crystal Growth and Polymorph Control of L-Glutamic Acid on Self-Assembled Monolayers

In this paper,we report a facile approach to obtain different polymorphs of L-glutamic acid(LGA) by crystallization on self-assembled monolayers of L-cysteine under different pH conditions.The β-glutamic acid crystals with plate-like shape were obtained at pH=2.0 and 2.5,while α-glutamic acid crystals were prepared at pH=3.0 and 4.0.The XRD results show that a small amount of β-glutamic acid crystals appear in α-glutamic acid crystals,and they are probably from the secondary nucleation of the β-form.