Characterization of ribostamycin and its impurities using a nano-quantity analyte detector: Systematic comparison of performance among three different aerosol detectors.

Characterization of aminoglycoside antibiotics like ribostamycin is important due to the complex composition and common toxic impurities. Aerosol detectors are often employed for determination of these non-absorbent analytes. In this work, a robust and cost-effective method was developed for simultaneous detection of ribostamycin and its related substances using high-performance liquid chromatography (HPLC) with a relative new aerosol detector named nano-quantity analyte detector (NQAD). With the introduction of less toxic but more compatible ion-pairs pentafluoropropionic acid (PFPA) and trifluoroacetic acid (TFA) in the eluent, an optimized separation effect was achieved. Compared with the other two aerosol detectors namely ELSD (evaporative light scattering detector) and CAD (charged aerosol detector), method verification and quantitative detection results revealed that NQAD had higher sensitivity than ELSD with a 0.8 µg/mL limit of detection, as well as wider linear range (from 2 µg/mL to 1000 µg/mL) than both CAD (from 2 µg/mL to 200 µg/mL) and ELSD (from 8 µg/mL to 200 µg/mL) detector. The performance of NQAD helped to realize detection of ribostamycin and its impurities with significant concentration differences in a single run. With a cation suppressor to eliminate the ion-suppression caused by the ion-pairs in the eluent, the structure of nine impurities in ribostamycin sample was characterized by liquid chromatography-mass spectrum (LC-MS). Both external standard and area normalization calculation were investigated, and NQAD obtained more accurate results due to its full-range linear response-to-concentration relationship, providing an alternative for routine quality control of multi analyte systems.

[Determination of fatty acid composition after saponification of common oil pharmaceutical excipients by supercritical fluid-evaporative light scattering method and its application in oil identification].

Oils and fats are commonly used in the pharmaceutical industry as solvents, emulsifiers, wetting agents, and dispersants, and are an important category of pharmaceutical excipients. Fatty acids with unique compositions are important components of oil pharmaceutical excipients. The Chinese Pharmacopoeia provides clear descriptions of the fatty acid types and limits suitable for individual oil pharmaceutical excipient. An unqualified fatty acid composition or content may indicate adulteration or deterioration. The fatty acid composition, as a key indicator for the identification and adulteration evaluation of oil pharmaceutical excipients, can directly affect the quality and safety of oil pharmaceutical excipients and preparations. Gas chromatography is the most widely used technique for fatty acid analysis, but it generally requires derivatization, which affects quantitative accuracy. Supercritical fluid chromatography (SFC), an environmentally friendly technique with excellent separation capability, offers an efficient method for detecting fatty acids without derivatization. Unlike other chromatographic methods, SFC does not use nonvolatile solvents (e. g., water) as the mobile phase, rendering it compatible with an evaporative light-scattering detector (ELSD) for enhanced detection sensitivity. However, the fatty acids in oil pharmaceutical excipients exist in the free and bound forms, and the low content of free fatty acids in these oil pharmaceutical excipients not only poses challenges for their detection but also complicates the determination of characteristic fatty acid compositions and contents. Moreover, the compositions and ratios of fatty acids are influenced by environmental factors, leading to interconversion between their two forms. In this context, saponification provides a simpler and faster alternative to derivatization. Saponification degrades oils and fats by utilizing the reaction between esters and an alkaline solution, ultimately releasing the corresponding fatty acids. Because this method is more cost effective than derivatization, it is a suitable pretreatment method for the detection of fatty acids in oil pharmaceutical excipients using the SFC-ELSD approach. In this study, we employed SFC-ELSD to simultaneously determine six fatty acids, namely, myristic acid, palmitic acid, stearic acid, arachidic acid, docosanoic acid, and lignoceric acid, in oil pharmaceutical excipients. Saponification of the oil pharmaceutical excipients using sodium hydroxide methanol solution effectively avoided the bias in the determination of fatty acid species and contents caused by the interconversion of fatty acids and esters. The separation of the six fatty acids was achieved within 12 min, with good linearity within their respective mass concentration ranges. The limits of detection and quantification were 5-10 mg/L and 10-25 mg/L, respectively, and the spiked recoveries were 80.93%-111.66%. The method proved to be sensitive, reproducible, and stable, adequately meeting requirements for the analysis of fatty acids in oil pharmaceutical excipients. Finally, the analytical method was successfully applied to the determination of six fatty acids in five types of oil pharmaceutical excipients, namely, corn oil, soybean oil, coconut oil, olive oil, and peanut oil. It can be combined with principal component analysis to accurately differentiate different types of oil pharmaceutical excipients, providing technical support for the rapid identification and quality control of oil pharmaceutical excipients. Thus, the proposed method may potentially be applied to the analysis of complex systems adulterated with oil pharmaceutical excipients.

Discrimination of polysorbate 20 by high-performance liquid chromatography-charged aerosol detection and characterization for components by expanding compound database and library.

Analyzing polysorbate 20 (PS20) composition and the impact of each component on stability and safety is crucial due to formulation variations and individual tolerance. The similar structures and polarities of PS20 components make accurate separation, identification, and quantification challenging. In this work, a high-resolution quantitative method was developed using single-dimensional high-performance liquid chromatography (HPLC) with charged aerosol detection (CAD) to separate 18 key components with multiple esters. The separated components were characterized by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) with an identical gradient as the HPLC-CAD analysis. The polysorbate compound database and library were expanded over 7-time compared to the commercial database. The method investigated differences in PS20 samples from various origins and grades for different dosage forms to evaluate the composition-process relationship. UHPLC-Q-TOF-MS identified 1329 to 1511 compounds in 4 batches of PS20 from different sources. The method observed the impact of 4 degradation conditions on peak components, identifying stable components and their tendencies to change. HPLC-CAD and UHPLC-Q-TOF-MS results provided insights into fingerprint differences, distinguishing quasi products.

An integrated liposome-based microfluidic strategy for rapid colorimetric analysis: A case study of microRNA-21 detection.

In this study, a novel integrated liposome-based microfluidic platform combined with a smartphone was designed for the rapid colorimetric detection of microRNA-21 (miRNA-21) in real samples. The flowing surface-functionalized liposomes were first captured by nucleic acid-functionalized Au nanoparticles in the microfluidic chip. In the presence of miRNA-21, the DNA strand modified on the surface of Au nanoparticles hybridized with the target to form double-stranded products and was cleaved by duplex-specific nuclease (DSN) enzyme, causing the liposomes to be re-released. Then, as the liposomes in the colorimetric module were lysed and the "cellular" contents were released, a step-by-step "glucose-glucose oxidase-3,3',5,5'-tetramethylbenzidine (TMB)" colorimetric reaction process catalyzed by the G-quadruplex/hemin was triggered. The grayscale values were recorded and recognized by the smartphone camera for miRNA-21 analysis. The advantages of the present strategy included the portability of smartphone-based colorimetric assay, the encapsulation and transport of reactants by liposomes and the low solvent usage of microfluidic chip. Under optimal conditions, this assay exhibited a wide linear range from 1 pM to 1 nM (r2 = 0.9981), and the limit of detection of miRNA-21 was as low as 0.27 pM. Moreover, the high specificity of this strategy allowed its successful application to the rapid analysis of miRNA-21 in real blood serum samples of people with type 2 diabetes.

Magnetic three-phase single-drop microextraction for highly sensitive detection of aflatoxin B1 in agricultural product samples based on peroxidase-like spatial network structure.

In this work, a highly sensitive method for aflatoxin B1 (AFB1) detection was developed based on a peroxidase-like spatial network structure. The specific antibody and antigen of AFB1 were coated on a histidine-modified Fe3O4 nanozyme to form the capture/detection probes. Based on the competition/affinity effect, the spatial network structure was constructed by the probes, which could be rapidly (8 s) separated by a magnetic three-phase single-drop microextraction process. In this single-drop microreactor, the network structure was applied to catalyze a colorimetric 3,3',5,5'-tetramethylbenzidine oxidation reaction for AFB1 detection. The signal was amplified significantly due to the strong peroxidase-like ability of the spatial network structure and the enrichment effect of the microextraction. Thus, a low detection limit (0.034 pg/mL) was achieved. The matrix effect of real sample can be eliminated by the extraction approach, and the practicability of this method was proved by agricultural product samples analysis.

Three-Dimensional Printed Microdevice to Enhance Headspace Microextraction for Enrichment of Histamine in Milk.

In this work, a three-dimensional (3D) printed microdevice was designed to fix a drop of extractant that was applied to the enrichment of the most toxic biogenic amine, histamine, by headspace single-drop microextraction (HS-SDME). Concomitantly, based on the hybridization chain reaction of the histamine aptamer isothermal nucleic acid amplification strategy, a new fluorescence sensing method was developed to realize the highly sensitive detection of histamine. This is the first application of a 3D-printed microdevice to realize the HS-SDME process, which, among other advantages, effectively solves the problem of unstable and variable drop volumes that can plague traditional SDME and ensures the accuracy and repeatability of the extraction process. The calibration linear range of this SDME-fluorescence method was from 10 pM to 5 µM (R2 > 0.98), and the limit of detection was as low as 3 pM. In addition, the method was successfully demonstrated to determine histamine spiked in milk, with recoveries of between 93% and 104%, and relative standard deviations of less than 5%. The method established in this study has important practical significance for food safety monitoring and human health and provides new ideas and solutions for the design and application of biosensors.

Ultrahigh-Performance Supercritical Fluid Chromatography and Detection of Multiple Biogenic Amines in Gentamicin Sulfate: Method Development Using Computer-Assisted Modeling.

In order to solve the problem of difficult separation of various biogenic amines (BAs), which have similar structures or very different polarities, in gentamicin, by conventional liquid chromatography, a new ultrahigh-performance supercritical fluid chromatography (UHPSFC) method was developed. In this method, 10 BAs were derivatized precolumn using dansyl chloride and separated using a UHPSFC system. By computational simulation, complete separation of 10 BAs was successfully achieved. Detection was performed using a photodiode array (PDA) and single-quadrupole mass spectrometry (MS) together with electrospray ionization (ESI). A wide linear range (10-2500 ng/mL) was achieved, with the limits of detection (LODs) between 1.2 and 10.0 ng/mL and the limits of quantification (LOQs) between 5.0 and 25.0 ng/mL. Apart from high sensitivity, this UHPSFC-PDA/ESI-MS detection method also displayed high accuracy, the matrix effect was reduced by an appreciable extent, and the recovery rates of the 10 BAs were between 84.1 and 117.1%. For comparison, high-performance liquid chromatography-tandem mass spectrometry (MS/MS) was also used for the detection of underivatized BAs in gentamicin, showing good linearity and high sensitivity (LODs from 0.05 to 1.00 ng/mL and LOQs from 1.00 to 12.50 ng/mL) for all BAs except for spermine and spermidine. Although single-quadrupole MS is inferior to MS/MS in terms of sensitivity, the UHPSFC method could detect more BAs. It also achieved the quantification limits required for impurity determination, demonstrating a potential strategy to offer a map overview of possible BA presence in fermentation antibiotics.

Magnetic Three-Phase Single-Drop Microextraction for Rapid Amplification of the Signals of DNA and MicroRNA Analysis.

The detection of nucleic acids usually suffers from a lengthy amplification process. To obtain an enhanced signal within several seconds, a magnetic three-phase single-drop microextraction (MTP-SDME) approach was developed for the quantification of nucleic acids. First, a target-triggered recycling amplification strategy was used to constitute magnetic branched DNA/Fe3O4 networks, which displayed peroxidase-like catalytic activity toward the 3,3',5,5'-tetramethylbenzidine colorimetric reaction. The networks were separated and enriched by rapid (6 s) MTP-SDME (with only 6 µL of solvent required), thereby producing highly sensitive signals for the quantification of nucleic acids. The signals were significantly amplified by the triple strategy (network formation, MTP-SDME, and catalytic reaction). The application of magnetic extraction minimized the background signal, avoided sample matrix effects, and enhanced the analyte signals. This assay achieved linear calibration curves of between 0.5 aM and 1 pM for microRNA-122 (miRNA-122) and between 1 aM and 1 pM for HBV-T (a DNA fragment from hepatitis B virus). Limits of detection of 0.15 aM for miRNA-122 and 0.34 aM for HBV-T were attained, with relative standard deviations of <5.0% (n = 3). Furthermore, the procedure was applied to determine miRNA-122 and HBV-T in genuine serum samples from hepatocellular carcinoma patients.

Temporal Sensing Platform Based on Bipolar Electrode for the Ultrasensitive Detection of Cancer Cells.

We report a bipolar electrode (BPE) sensing platform for the temporal detection of cancer cells. Combining the advantages of anodic dissolution and electrochemiluminescence (ECL), this strategy shows an ultralow detection limit down to 5 cells/cm(2). At the anode working as the reporting pole, Au NPs were assembled through DNA double strand, which served as both catalyzer for the ECL reaction of luminol/H2O2 and seeds for the chemical reduction of Ag, the anodic dissolution probe. The duration of Ag layer dissolution was positively correlated with the amount of Ag but negatively related to the controlled potential and the conductivity of the circuit. Therefore, it was possible to amplify a slight conductivity change through tuning the other two factors. As the formation of Ag@Au completely quenched the ECL emission of luminol, the ECL emission recovery reflected the extent of anodic dissolution. Through monitoring the ECL recovery time before and after the incubation of cells on the cathode, a few number of cells could be quantified due to slight difference of the conductivity. This method shows several merits. First, the combination of anodic dissolution and ECL significantly increases the detection sensitivity of BPE device. In addition, this strategy broadens the application of BPE for the ultrasensitive monitoring of cancer cells, which was applied to investigate the capture efficiencies of antibodies and aptamers toward MCF-7 and A549.

Joint enhancement strategy applied in ECL biosensor based on closed bipolar electrodes for the detection of PSA.

A highly sensitive electrogenerated chemiluminescence (ECL) biosensor was developed on the basis of a closed bipolar electrode (BPE) apparatus for the analysis of prostate specific antigen (PSA). Bipolar modifications bring up two different stages of enhancement on the same electrode. Anodic enhancement was conducted by modifying gold nanoparticles (Au NPs) to catalyze the anodic ECL reaction between luminol and hydrogen peroxide. Cathodic introduction of thionine tagged PSA antibody led to a further pertinently enhancement synchronized with the PSA amount variation, because the existence of thionine greatly increased the rate of electron gains on cathode, leading to the corresponding acceleration of anodic ECL reaction. The more thionine modified target molecules were introduced, the faster luminol was oxidized, the higher faraday current approached, and sensitive quantification was realized in correlation with the responsive ECL intensity differences. The quantification resulted in a good determination range between 0.1pg/mL and 0.1µg/mL. This strategy mainly took advantage of the special structure of closed BPE to realize a simultaneous amplification on both ends of BPE. Moreover, the platform had a potential of providing a multi-functional strategy for the realization of other bio-detections by simply substituting the PSA sandwich structure with other bio-structures.

A ratiometric electrochemiluminescence detection for cancer cells using g-C3N4 nanosheets and Ag-PAMAM-luminol nanocomposites.

In this work, a dual-signaling electrochemiluminescence (ECL) ratiometric sensing approach for the detection of HL-60 cancer cells was reported for the first time. G-C3N4 nanosheets and Ag-PAMAM-luminol nanocomposits (Ag-PAMAM-luminol NCs) were prepared and served as reductive-oxidative and oxidative-reductive ECL emitters respectively. DNA probe functionalized Ag-PAMAM-luminol NCs would hybridize with aptamers modified onto magnetic beads. In the presence of HL-60 cells, the aptamer would conjugate with the target cell and release Ag-PAMAM-luminol NCs. After magnetic separation, released Ag-PAMAM-luminol NCs would hybridize with capture DNA on g-C3N4 nanosheets. ECL from g-C3N4 nanosheets coated on ITO electrode at -1.25 V (vs SCE) could be quenched by Ag-PAMAM-luminol NCs due to the resonance energy transfer (RET) from g-C3N4 nanosheets to Ag NPs. Meanwhile, Ag-PAMAM-luminol brought the ECL signal of luminol at +0.45 V (vs SCE). Thus, the concentration of HL-60 cancer cells could be quantified by both the quenching of ECL from g-C3N4 nanosheets and the enhancement of ECL from luminol. By measuring the ratio of ECL intensities at two excitation potentials, this approach could achieve sensitive and reliable detection for cancer cells in a wide range from 200 cells/mL to 9000 cells/mL with the detection limit of 150 cells (S/N=3).

General Strategy for Enhancing Electrochemiluminescence of Semiconductor Nanocrystals by Hydrogen Peroxide and Potassium Persulfate as Dual Coreactants.

Semiconductor nanocrystals usually suffer from weak electrogenerated chemiluminescence (ECL) emissions compared with conventional organic emitters. In this work, we propose, for the first time, a very convenient but effective way to greatly enhance ECL emission of semiconductor TiO2 nanotubes (NTs) by H2O2 and K2S2O8 as dual coreactants, generating ECL emission ca. 6.3 and 107 times stronger than that of K2S2O8 or H2O2 as an individual coreactant, respectively. Scanning electron microscopy, X-ray diffraction, and electron paramagnetic resonance spectral studies were carried out to investigate the ECL enhancement mechanism. The ECL enhancement of TiO2 NTs by the K2S2O8-H2O2 system was supposed to originate from the coordination of H2O2 to the TiO2 surface and the synergy effect between H2O2 and K2S2O8 in the ECL process. The coordination of H2O2 to the surface of TiO2 could stabilize the electrogenerated coreactant-related radical OH(•) (hydroxyl radical), which could obviously promote the amount of sulfate radical anion (SO4(•-)) near the electrode surface by inducing decomposition of K2S2O8 into SO4(•-) or inhibiting the consumption of SO4(•-) by its reaction with H2O. The holes (h(+)) released from SO4(•-) were injected into the valence band of TiO2, resulting in more TiO2(+), which combined with the electrons coming from the conduction band with an enhanced light emission. Moreover, this enhancement effect was also applicable to ECL of a CdS nanocrystal film on a glass carbon electrode, with ca. 2.74- and 148.3-fold enhanced ECL intensity correspondingly, indicating wide applications in the development of semiconductor nanocrystal-based ECL biosensors.

Visual electrochemiluminescence detection of cancer biomarkers on a closed bipolar electrode array chip.

This paper describes a novel electrochemiluminescence (ECL) imaging platform for simultaneous detection of cancer biomarkers based on a closed bipolar electrode (BPE) array. It consists of two separated channel arrays: detection channel array and sensing channel array, which are connected by a group of parallel ITO BPEs on a glass substrate. Besides, two parallel ITO strips are fabricated at the two sides of BPE array and employed as driving electrodes. After Au films are electrochemically deposited on the cathodes of the BPE array, nanobioprobes including biorecognition elements (aptamer or antibody) and a novel electrochemical tag, which is synthesized by doping thionine in silica nanoparticles (Th@SiO2 NPs), are introduced into the cathodes by immunoreaction or DNA hybridization. The Th@SiO2 coupled nanobioprobes as both recognition probes and signal amplification indicators could mediate the ECL signals of Ru(bpy)3(2+)/tripropylamine (TPA) on the anodes of BPE array through faradaic reaction due to the charge neutrality of BPE. Thus, multiplex detection of cancer biomarkers (adenosine triphosphate (ATP), prostate-specific antigen (PSA), α-fetoprotein (AFP) and thrombin) is realized by forming specific sensing interfaces onto the cathodic poles of BPEs in different sensing channels and reported by the ECL images of the Ru(bpy)3(2+)/TPA system on the anodic poles of BPEs in detection channels. The results demonstrate that this visual ECL platform enables sensitive detection with excellent reproducibility, which may open a new door toward the development of simple, sensitive, cost-effective, and high throughput detection methods on biochips.

Visual electrochemiluminescence detection of telomerase activity based on multifunctional Au nanoparticles modified with G-quadruplex deoxyribozyme and luminol.

A novel visual electrochemiluminescence (ECL) analysis strategy for detection of telomerase activity is reported on a microarray chip, with G-quadruplex deoxyribozyme (DNAzyme) and luminol modified Au nanoparticles (NPs) as double-catalytic amplification labels.

Electrochemiluminescence aptasensor based on bipolar electrode for detection of adenosine in cancer cells.

Here we report a novel approach for the detection of adenosine in cancer cells by electrochemiluminescence (ECL) on a wireless indium tin oxide bipolar electrode (BPE). In this approach, ferrocene (Fc) which is labeled on adenosine aptamer is enriched on one pole of the BPE by hybridization with its complementary DNA (ssDNA) and oxidized to Fc(+) under an external voltage of 5.0V at the two ends of BPE. Then, a reversed external voltage was added on the BPE, making Fc(+) enriched pole as cathode. The presence of Fc(+) promotes the oxidation reaction on the anodic pole of the BPE, resulting in a significant increase of ECL intensity using Ru(bpy)3(2+)/tripropylamine (TPA) system as test solution. The presence of target adenosine was reflected by the ECL signal decrease on the anodic pole caused by the target-induced removal of ferrocene-aptamer on the cathodic pole. The decrease of ECL signal was logarithmically linear with the concentration of ATP in a wide range from 1.0 fM to 0.10 µM. This ECL biosensing system could accurately detect the level of adenosine released from cancer cells.

Microchip device with 64-site electrode array for multiplexed immunoassay of cell surface antigens based on electrochemiluminescence resonance energy transfer.

This paper describes a novel on-chip microarray platform based on an electrochemiluminescence resonance energy transfer (ECL-RET) strategy for rapid assay of cancer cell surface biomarkers. This platform consists of 64 antigen-decorated CdS nanorod spots with the diameter of 1.0 cm uniformly distributed on 16 indium tin oxide (ITO) strips, which is coated with a multichannel decorated polydimethylsiloxane (PDMS) slice to realize multiplexed determination of antigens. To shorten the immune reaction time in the microchannels and simplify the device, magnetic stirring and four-channel universal serial bus (USB) ports for plug-and-play were used. When Ru(bpy)(3)(2+) labeled antibodies were selectively captured by the corresponding antigens on the CdS nanorod spot array, ECL-RET from the CdS nanorod (donor) by cathodic emission in the presence of K(2)S(2)O(8) to Ru(bpy)(3)(2+) (acceptor) occurred. With signal amplification of Ru(bpy)(3)(2+) and competitive immunoassay, carcinoembryonic antigen (CEA), α-fetoprotein (AFP), and prostate specific antigen (PSA) as models were detected on this microfluidic device via recording the increased ECL-RET signals on electrode surfaces. Furthermore, this multiplexed competitive immunoassay was successfully used for detecting cancer cell surface antigens via the specific antibody-cell interactions and cell counting via cell surface receptors and antigens on the CdS nanorod surface. This platform provides a rapid and simple but sensitive approach with microliter-level sample volume and holds great promise for multiplexed detection of antigens and antigen-specific cells.

Electrochemiluminescence analysis of folate receptors on cell membrane with on-chip bipolar electrode.

In this paper we report a transparent bipolar electrode based microfluidic chip-electrochemiluminescence (ECL) system for sensitive detection of folate receptors (FR) on cell membranes. This integrated system consists of a poly(dimethylsiloxane) (PDMS) layer containing a microchannel and a glass bottom sheet with indium tin oxide (ITO) strips as bipolar detectors. The ITO strips are fabricated using a PDMS micromold with carbon ink as a protective layer in place of traditional photoresist. The configuration of the bipolar electrode has great influence on the ECL intensity of Ru(bpy)(3)(2+)/tripropylamine(TPA) system. Further studies show that folic acid (FA) can strongly inhibit the ECL of the Ru(bpy)(3)(2+)/TPA system. Based on specific recognition between FA and FR on cell membrane, this microfluidic chip-ECL system is successfully applied for detecting the level of FR on human cervical tumor (HL-60) cells and MEF cells. It is found that the ECL intensity increases with the number of HL-60 cells in the range of 21 to 3.28 × 10(4) cells/mL. The average level of FR on HL-60 cells is calculated to be 8.05 ± 0.75 × 10(-18) mol/cell. While for MEF cells, it shows a much slower ECL increment than HL-60 cells due to the much lower FR level on MEF cells (5.30 ± 0.61 × 10(-19) mol/cell). Moreover, exocytosis of FA after FR mediated endocytosis was observed according to the change of the ECL signal with the incubation time of HL-60 cells in the FA- Ru(bpy)(3)(2+)/TPA system.

CdS quantum dots/Ru(bpy)3(2+) electrochemiluminescence resonance energy transfer system for sensitive cytosensing.

In this paper, an electrochemiluminescence resonance energy transfer (ECL-RET) system from CdS quantum dot to Ru(bpy)(3)(2+) was developed for the first time. By the signal amplification of Ru(bpy)(3)(2+) and the specific antibody-cell surface interactions, this ECL-RET system could sensitively respond down to 12.5 SMMC-7721 cells per mL.