D-alanyl-D-alanine-Modified Gold Nanoparticles Form A Broad-Spectrum .
Theranostics 2018, Vol. 8, Issue 5IvyspringInternational PublisherResearch Paper1449Theranostics2018; 8(5): 1449-1457. doi: 10.7150/thno.22540D-alanyl-D-alanine-Modified Gold Nanoparticles Form aBroad-Spectrum Sensor for BacteriaXinglong Yang1,2,3,5, Yan Dang4, Jinli Lou4, Huawu Shao2 , and Xingyu Jiang1, 3 1.2.3.4.5.CAS Center for Excellence in Nanoscience, CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, Beijing Engineering Research Center forBioNanotechnology, National Center for NanoScience and Technology, ZhongGuanCun BeiYiTiao, Beijing, 100190, ChinaNatural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, 610041, ChinaUniversity of Chinese Academy of Science, Beijing, 100049, ChinaDepartment of Clinical Laboratory, Beijing You’an Hospital Affiliated to Capital Medical University, No. 8 You’an Men Wai Xi TouTiao, Fengtai District,Beijing, 100069, ChinaPresent address: School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China Corresponding author: Xingyu Jiang, Email: xingyujiang@nanoctr.cn, Tel.: 86-10-82545620; Huawu Shao, Email: shaohw@cib.ac.cn, Tel.: 86-028-82890818 Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) /4.0/). See http://ivyspring.com/terms for full terms and conditions.Received: 2017.08.25; Accepted: 2017.12.23; Published: 2018.02.04AbstractRationale: Rapid and facile detection of pathogenic bacteria is challenging due to the requirementof large-scale instruments and equipment in conventional methods. We utilize D-amino acid asmolecules to selectively target bacteria because bacteria can incorporate DADA in its cell wall whilemammalian cells or fungi cannot.Methods: We show a broad-spectrum bacterial detection system based on D-amino acid-cappedgold nanoparticles (AuNPs). AuNPs serve as the signal output that we can monitor without relyingon any complex instruments.Results: In the presence of bacteria, the AuNPs aggregate and the color of AuNPs changes fromred to blue. This convenient color change can distinguish between Staphylococcus aureus (S. aureus)and methicillin-resistant Staphylococcus aureus (MRSA). This system can be applied for detection ofascites samples from patients.Conclusion: These D-amino acid-modified AuNPs serve as a promising platform for rapid visualidentification of pathogens in the clinic.Key words: gold nanoparticles, D-amino acid, bacteria, spontaneous bacterial peritonitis, colorimetric biosensorIntroductionSpontaneous bacterial peritonitis (SBP) causedby ascites is a common cause of death in patientssuffering from cirrhosis.[1, 2] The fundamentalchallenge in mitigating SBP is the early detection ofthese bacteria in ascites. The current standards fordetecting bacteria are microbial culture or geneanalysis, which need complex equipment andprofessional technicians to operate in specificenvironments. Recent studies show that colorimetricbiosensors, fluorescence imaging or microelectronicscan be useful for Gram-positive and Gram-negativebacteria detection.[3-12]AuNPs are widely applicable as colorimetricbiosensors, drug delivery carriers, antibacterialagents, and photothermal therapy material.[13-28]Our group has developed a series of sistant bacteria.[29-31] We also report anumber of colorimetric sensors based on AuNPs sincetheir use can avoid the necessity of bulky instrumentsby obtaining signals with the naked eye.[3, 32-36] Anumber of investigators have reported bacterialbiosensors based on AuNPs. Scientists oated AuNPs to detect bacteria.[37] CTAB ispositively charged such that it can target thehttp://www.thno.org
Theranostics 2018, Vol. 8, Issue 1negatively charged bacterial surface. Researchers alsouse antibodies or aptamers to decorate AuNPs forbacterial targeting.[38] Unfortunately, these strategiesare based on charge interactions and easily interferedby surroundings such as pH values, metal ion orproteins. A stable, easily available and navigableplatform for the detection of pathogens is urgentlyneeded.We hypothesize that D-amino acids can play arole in AuNPs-based detection of microorganismsbecause they mainly exist in bacteria. In thesemicroorganisms, D-amino acids are part ofpeptidoglycan, the principal component of thebacterial cell wall, which surrounds bacteria.[39, 40]Given the critical role of peptidoglycan during thesynthesis of cell wall and bacterial reproduction,researchers have developed a number of antibioticsthat can obstruct peptidoglycan synthesis to inhibitbacterial growth.[41] Recently, researchers havefound that a number of bacteria can easily incorporateexogenous D-amino acids from the surroundingmedium into their peptidoglycan via displacing theterminal D-alanine of the oligopeptide strand, due dases.[42, 43] They used D-aminoacids linked with fluorescent molecules to specificallylabel the peptidoglycan network.[44-46] However,these imaging methods are inconvenient and theymostly rely on complex instruments to label anddetect bacteria. We thus wonder if we can modify the1450surface of AuNPs with D-amino acids to diagnosebacterial infection.Here, we illustrate a novel strategy for -D-alanine, DADA) with AuNPs. AuNPsserve as the signal output that we can monitorwithout relying on any complex instruments. Weutilize DADA as molecules to selectively targetbacteria because bacteria can incorporate DADA in itscell wall while mammalian cells or fungi cannot.[47]When bacteria consume DADA capped on AuNPs,DADA unbinds from the AuNP surface, and theAuNPs become unstable and aggregate becauseDADA no longer protects the surface of the AuNPs.We prepared DADA-modified AuNPs (Au DADA)in a one-pot reaction without any other complexmodifications (Scheme 1). We employed the L-aminoacid, L-Alanyl-L-alanine (LALA), as comparison toconfirm the hypothesized mechanism of interactionbetween bacteria and Au DADA. We choselaboratory antibiotic-sensitive bacteria and clinicalmultidrug-resistant (MDR) bacteria to evaluate thedetection activity of our AuNPs. Interestingly,Au DADA could distinguish between S. aureus andMRSA. To investigate the potential of Au DADA inclinical application, we used it to diagnose bacterialinfection in liver ascites from patients. Our studypaves a new way to colorimetric biosensing ofpathogens.Scheme 1. Schematic illustration of the synthesis of D-amino acid-modified AuNPs and the structural change of peptidoglycan after incubation with D-aminoacid-modified AuNPs.http://www.thno.org
Theranostics 2018, Vol. 8, Issue 1Materials and MethodsMaterialsGold (III) chloride hydrate rohydride are from Aladdin Industrial Corporation(China). L-Alanyl-L-alanine (LALA) is from Sigma.We used the procured chemicals without furtherpurification. We used a Milli-Q purification system toobtain Deionized (DI) water (18.2 MΩ·cm). Weobtained zeta potential values of AuNPs withZetasizer Nano ZS (Malvern Instruments). Weobtained the ascites samples from Beijing You’anHospital with patients’ consent and approval by thelocal Ethics Committee.Preparation of AuNPsWe stirred the mixture of D-amino acids (DADAor LALA) (16 mg, 0.1 mmol, dissolved in 6 mL of DIwater, 50 μL of absolute acetic acid and 20 mg ofTween 80) and HAuCl4.3H2O (0.1 mmol, dissolved in0.5 mL DI water) for 10 min and add NaBH4 (0.3mmol freshly dissolved in 2.5 mL DI water) dropwisewith vigorous stirring. The color of the mixturechanged to dark red immediately. We kept stirring themixture for 1 h at 0 C. We dialyzed (14 kDa MWcutoff, Millipore) it for 48 h with DI water, sterilized itthrough a 0.22 μm filter (Millipore), and stored it at 4 C for use. We synthesized different ratios ofAu DADA using the same method.Characterization of AuNPsWe investigated the morphologies of AuNPs viatransmission electron microscopy (TEM) (Tecnai G220 ST TEM) from the American FEI company. Amicroplate reader (Tecan infinite M200) gave usultraviolet-visible (UV-vis) spectra. We prepared TEMsamples by dropping 5 μL of the samples onformvar/carbon coated copper grids and dried themovernight. We measured zeta potential using MalvernZetasizer.Bacteria cultureWe cultured bacteria in Luria-Bertani (LB) brothmedium (5 g/L NaCl, 10 g/L tryptone powder, and 5g/L beef extract powder, pH 7.2) at 37 C on a shakerbed at 200 rpm for 4 h. Then, we diluted bacteria withLB broth to a concentration of 1.0 108 CFU/mL,which corresponded to an optical density of 0.1 at 600nm measured by UV-vis spectroscopy.Identification of bacteriaWe pre-cultured Staphylococcus aureus (S. aureus,ATCC 6538P), MRSA, E. coli (ATCC 11775), MDR E.coli, Klebsiella pneumoniae (K. pneumonia, ATCC 13883),1451MDR K. pneumoniae, Bacillus subtilis (B. subtilis, ATCC66633), and Listeria monocytogenes (L. monocytogenes,ATCC 19115) in LB broth to a concentration of 1.0 108 CFU/mL. We diluted them to a concentration of1.0 106 CFU/mL. We performed the assay foridentification of bacterial strains in 96-wellmicroplates (Constar, 3599). We added 90 µL AuNPsdiluted with LB medium to the plate. Then we added10 µL bacteria prepared in the AuNPs medium (finalconcentration: 1.0 105 CFU/mL). We used AuNPs asa control group. Each group has 3 replicates. In orderto keep bacteria in good form, we incubated them at37 C in an incubator.The cytotoxicity of Au DADAWe employed Cell Counting Kit-8 (CCK-8) tomeasure the cytotoxicity of Au DADA. We usedDulbecco’s modified Eagle’s medium (DMEM; Gibco)containing fetal bovine serum (FBS, 10%; Gibco),penicillin and streptomycin (1%), and glutamine (1%)to culture human umbilical vein endothelial cells(HUVECs) and human cervical cancer (HeLa) cellsand incubated in CO2 (5%) at 37 C. HUVEC cellswere grown overnight on 96-well culture plates ( 10,000 cells per well) and then we added varyingconcentrations of Au DADA to the 96-well plate.After 24 h incubation, we used culture medium towash cells and stained them with 10 μL of CCK perwell. We measured the optical density of the cells at450 nm by a microplate reader (Tecan infinite M200)after incubating for 2 h.Stability of Au DADAWe utilized various pH values ranging frompH 1 to pH 14 to study the stability of Au DADA.We used 3 M HCl and 2 M NaOH to obtain the pHsolutions. We incubated Au DADA in these solutionsfor 4 h.Results and DiscussionSynthesis and characterization of Au DADAWe prepared Au DADA through a one potprocess via reduction of tetrachloroauric acid bysodium borohydride in the presence of DADAmolecules in deionized (DI) water. The role of DADAin this reaction is to stabilize and disperse the NPs.Our synthetic strategy is straightforward andconvenient without other chemical modifications.Diameters of spherical Au DADA are around 6 nm asindicated by TEM images and statistical analysis(Figure 1A). UV-vis spectra reveal the plasmonresonance of NPs at 520 nm (Figure 1B). We testedzeta potential values of the NPs in water at 25 C. Theresults show that Au DADA are negatively charged(–21.9 mV), which is the same as the charges ofhttp://www.thno.org
Theranostics 2018, Vol. 8, Issue 11452bacterial surfaces (Table S1). We used FourierTransform Infrared Spectroscopy (FT-IR) to furthercharacterize the AuNPs (Figure 1C). The asymmetricstretching mode of N-H has a band with a maximumat 3334 cm-1 both in pure DADA and DADA-cappedAuNPs, which confirms the presence of DADA onAuNPs. The bending mode of N-H at 1720 cm-1 and1677 cm-1 can further verify the results.monocytogenes) and clinical MDR isolates (MRSA,MDR E. coli, MDR K. pneumoniae). TheseGram-positive (S. aureus, B. subtilis, L. monocytogenes,MRSA) and Gram-negative (E. coli, K. pneumonia,MDR E. coli, MDR K. pneumoniae) bacteria arecommon in clinical ascites samples. We addedAu DADA to microplate wells and mixed with LBbroth (as control) and different types of bacteria withthe same concentration. The color of the media fromcells that contain bacteria changed to blue or purple,Colorimetric detection of bacteria viawhereas the color of the control group remained redAu DADA(Figure 2B). We can visually distinguish samples thatIn order to obtain AuNPs for sensitive bacterialhave bacteria from those that do not. We found thatdetection, we investigated the effect of Au DADAthe well containing MRSA turned purple instead ofconcentration on the colorimetric response. Weblue like the other species. This difference may beincubated S. aureus with various concentrations ofcaused by the different activity of penicillin bindingAu DADA (0.5, 2.5, 5, 10, 15, 20, and 25 μM). Weproteins (PBPs), which influences transpeptidationfound that the groups of 0.5, 2.5, and 5 μM are moreand synthesis of peptidoglycan.[48] This observationsensitive than other groups (Figure S1). Because thecan potentially distinguish between S. aureus andcolor change at 5 μM is the most significant and can beMRSA.read out easily, we chose it for subsequentTo quantify the colorimetric response of AuNPs,experiments.we tested the absorption spectra of each AuNPsWe tested the colorimetric response ofsolution in the presence of bacteria (Figure 2A). AllAu DADA by incubating with various types ofsamples show a red-shift in their absorption spectrabacteria, including laboratory antibiotic-sensitiveafter addition of bacteria. The observations from thestrains (S. aureus, E. coli, K. pneumoniae, B. subtilis, L.spectra are consistent with the visual observations,where the AuNPs in all wellsunderwent a color change from redto blue. The absorbance at 520 nmdecreased and the absorbance at600 nm increased. We used theratio between the absorbancevalues of AuNPs at 600 nm and 520nm (A 600 nm/A 520 nm) to evaluatethe degree of AuNPs aggregation(Figure 2B). A low A 600 nm/A 520 nmindicates that AuNPs disperse wellin the solution, and a high A 600nm/A 520 nm indicates aggregation ofAuNPs. All samples with bacteriaare different from the controlsolution without bacteria. The onlyoutstanding sample is MRSA,whose A 600 nm/A 520 nm is lowerthan the A 600 nm/A 520 nm of S. aureusand other bacterial solutions. Thisobservation correlates well with thepurple color of MRSA. Despite thisdifference, A 600 nm/A 520 nm ofMRSA is still significantly higherthan A 600 nm/A 520 nm of pureAuNPs. These facts confirm thatAu DADA can indeed detect bothFigure 1. Characterization of Au DADA. (A) TEM image of nanoparticles. Inserted structure is DADA. (B)Gram-positive and Gram-negativeAbsorption spectrum of Au DADA. Inserted photo is Au DADA in water. (C) FT-IR of Au DADA andDADA.bacteria.http://www.thno.org
Theranostics 2018, Vol. 8, Issue 11453Figure 2. Response of Au DADA to different types of bacteria. (A) Absorption spectra of Au DADA incubated with bacteria. (B) Change in color of Au DADAcaused by varying types of bacteria and the plot of A 600 nm/A 520 nm of AuNPs versus different types of bacteria. Mean values and standard deviations were obtainedfrom five independent experiments.Figure 3. The sensitivity of Au DADA for detection of S. aureus. (A) A 600 nm/A 520 nm value of Au DADA. (B) The linear relationship between the A 600 nm/A 520 nmvalue and the concentration of S. aureus. Mean values and standard deviations were obtained from five independent experiments.We employed S. aureus as an example to studythe sensitivity of our system. We exposed Au DADAto the S. aureus solutions with different concentrations(1 102-1 108 CFU/mL). When the concentration ofbacteria is high, the value of A 600 nm/A 520 nm is high,indicating the high sensitivity of Au DADA to highconcentrations of bacteria (Figure 3). The limit ofdetection (LOD) was evaluated using the formula:LOD 3S/M, where S represents the value of thestandard deviation of blank samples and M is theslope of the standard curve within the concentrationrange. According to the formula, the LOD ofAu DADA is 3.2 102 CFU/mL. The limit ofquantitation (LOQ, LOQ 10S/M) is 1.1 103CFU/mL. These results show that Au DADA hasrelatively high sensitivity.TEM characterization of interactions betweenbacteria and Au DADAWe employed TEM to characterize Au DADAaggregation on bacteria. We used S. aureus as anexample to perform this assay (Figure 4 and S2).Au DADA aggregated around bacterial surfaces. Incomparison, the TEM of the control groups show thatthere were no AuNPs around bacteria and Au DADAitself did not aggregate without bacteria (Figure 4Band 4C).The mechanism of aggregationIt is evident from other studies that bacteria canincorporate exogenous D-amino acids into theirpeptidoglycan. Thus, we speculate that the D-aminoacid is critical for the aggregation of AuNPs in ourhttp://www.thno.org
Theranostics 2018, Vol. 8, Issue 1study. We prepared L-amino acid modified-AuNPs(Au LALA) to confirm this hypothesis. The syntheticmethod for Au LALA is the same as for Au DADApreparation. TEM and UV-vis spectra of Au LALAdemonstrate that they are similar to Au DADA(Figure S3). We used S. aureus and E. coli to test theability of bacterial detection about Au LALA (FigureS4A and S4B). Au LALA had no response to S. aureusand E. coli even when we cultured them for 16 h. Thisobservation agrees with literature reports showingthat bacteria can only incorporate exogenous D-aminoacids.To further investigate the relationship betweenbacterial peptidoglycan and D-amino acids, weselected C. albicans, a kind of fungi, whose cell wallbiosynthesis does not involve peptidoglycan and thuscannot incorporate D-amino acid (Figure S4C andS4D).[49, 50] We cultured C. albicans with Au DADAin potato dextrose agar (PDA) broth in 96-well platesat 25 C. We recorded the optical density of thesemixtures at different time periods via a microplatereader (Tecan infinite M200). The color of Au DADAdid not change when incubating with C. albicans foreven 36 h. This observation further demonstrates thatthe aggregation of Au DADA is due to incorporationof D-amino acid.The zeta potential of Au DADA is –21.9 mV.However, bacterial surfaces are also negative charged.Therefore, it is reasonable to deduce that there isnegligible electrostatic interaction between positivelycharged Au DADA and negatively charged bacterialsurface.1454We employed FT-IR to further characterize theaggregated Au DADA after incubating with bacteria(Figure S5). The characteristic peaks of theasymmetric stretching mode of N-H (3334 cm-1) andthe bending mode of N-H at 1720 cm-1 and 1677 cm-1disappeared compared with DADA and the originalAu DADA, which confirms the reduction of surfacedensity of DADA on AuNPs.These observations lead us to deem thatD-amino acid-modified AuNPs became unstable andaggregated after bacteria consumed the D-amino acidcapped on AuNPs.Cytotoxicity of Au DADAAs biological sensors, the low cytotoxicity ofmaterials is important. Thus, we set out to evaluatethe toxicity of Au DADA. We incubated HUVECsand HeLa cells in the presence of differentconcentrations of Au DADA for 24 h and analyzedthem by CCK-8. We observed no obvious loss ofviability of both HUVECs and HeLa cells, whichhighlights the outstanding biocompatibility ofAu DADA (Figure S6).Stability of Au DADAWe tested the stability of Au DADA at differentpH values (pH 1-14) (Figure S7). We put Au DADAin aqueous solutions of different pH for 4 h. Thecolors of Au DADA in aqueous solutions of differentpH are essentially the same (Figure S7A). The UV-visspectra of Au DADA at various pH levels are alsoessentially the same (Figure S7B).Figure 4. TEM images of S. aureus and Au DADA. (A) S. aureus incubated with Au DADA. (B) S. aureus by itself. (C) Morphology of Au DADA. The insertedimage is the full view of the bacterium.http://www.thno.org
Theranostics 2018, Vol. 8, Issue 11455Figure 5. Bacterial detection of ascites samples. (A) Absorption spectra and photos of Au DADA incubated with bacteria. (B) The plot of A 600 nm/A 520 nm of AuNPsversus different types of bacteria in ascites samples. Mean values and standard deviations were obtained from five independent experiments.To further test the selectivity of our assay, weadded FeCl3, CaCl2, CuCl2, BaCl2, Hg(ClO4)2, AgNO3,NaCl, KCl into Au DADA solution at a finalconcentration of 500 μM and stored at 37 C for 8 h.Au DADA did not aggregate according to the UV-visspectra of Au DADA. This result demonstrates thehigh selectivity of Au DADA (Figure S8).We evaluated the stability of Au DADA storedfor almost 15 months at 4 C and for 2 months at 37 Cand found them to be well dispersed (Figure S9).These results demonstrate that Au DADA hasoutstanding stability even in highly acidic or alkalineenvironments.band at 520 nm decreased, along with peakbroadening and distinctive red-shift, and a broadabsorption at 600 nm emerged (Figure 5A). Weemployed the ratio between the absorbance values ofAuNPs at 600 nm and 520 nm (A 600 nm/A 520 nm) to testthe degree of AuNP aggregation. The ratio of A 600nm/A 520 nm is also a function of the presence of bacteriain clinical samples (Figure 5B). We also tested if thematrix of ascites affects the detecting system. Theascitic fluid had no effect to our assay, which showsthe highly stability of Au DADA (Figure S8). Theseresults further validate the potential applications inbacterial monitoring.Applicability of the detection system in a realclinical settingConclusionsTo evaluate the applicability of this detectionsystem, we applied the method to detect bacteriawithin ascites samples from patients (Figure 5). Weincubated Au DADA with the clinical samples ofascites that have pathogens such as S. enotrophomonas maltophilia (S. maltophilia), orPseudomonas aeruginosa (P. aeruginosa). Because thesebacteria are clinically important, we explored thegenerality of our approach by expanding beyond thetypes of bacteria we used in the model experiments.The color of Au DADA changed after incubating withdifferent kinds of pathogens compared with thecontrol group (Au DADA media). When pathogensexisted in the samples, the color of Au DADAchanged from red to blue. The absorption spectra ofthe samples have obvious changes. The absorbanceWe report a versatile and stable platform basedon D-amino acid-coated AuNPs for colorimetricdetection of bacteria that can also distinguish betweenS. aureus and MRSA. This is the first report thatcombines D-amino acid with AuNPs to detectbacteria. Although the sensitivity of this system is notas high as reported methods and it is abroad-spectrum sensor for bacteria, its stability issuperior. We believe that Au DADA is a promisingbiosensor for simple detection of bacterialcontaminants for clinical diagnostics. We also openthe way to bacterial theranostics using D-amino acidand ;S. aureus:Staphylococcus aureus; MRSA: methicillin-resistantStaphylococcus aureus; SBP: spontaneous bacterialhttp://www.thno.org
Theranostics 2018, Vol. 8, Issue 1peritonitis; CTAB: cetyltrimethylammonium bromide;DADA: D-alanyl-D-alanine; LALA: L-Alanyl-Lalanine; MDR: multidrug-resistant; DI: Deionized;TEM: transmission electron microscopy; UV-vis:ultraviolet-visible; LB: Luria-Bertani; K. pneumonia:Klebsiella pneumoniae; B. subtilis: Bacillus subtilis; L.monocytogenes: Listeria monocytogenes; CCK-8: CellCounting Kit-8; DMEM: Dulbecco’s modified Eagle’smedium; FBS: fetal bovine serum; HUVECs: humanumbilical vein endothelial cells; HeLa: human cervicalcancer; LOD: the limit of detection; LOQ: the limit ofquantitation; PDA: potato dextrose agar; FT-IR:Fourier Transform Infrared Spectroscopy.Supplementary MaterialSupplementary figures and mentsWe thank the Ministry of Science andTechnology of China (2013YQ190467), ChineseAcademy of Sciences (XDA09030305) and theNational Science Foundation of China (81361140345,51373043, 21535001) for financial support.Competing InterestsThe authors have declared that no competinginterest g Y, Lou J, Yan Y, Yu Y, Chen M, Sun G. et al. 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Key words: gold nanoparticles, D-amino acid, bacteria, spontaneous bacterial peritonitis, colorimetric biosensor Introduction Spontaneous bacterial peritonitis (SBP) caused by ascites is a common cause of death in patients suffering from cirrhosis.[1, 2] The fundamental challenge in mitigating SBP is the early detection of
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High purity (99.9% AR grade) samples of L alanine, potassium chloride and potassium iodide were used for the crystal preparation. The saturated solutions of L alanine and potassium chloride were prepared using doubly di
USP Dietary Supplements Reference Standards Catalog Page 2 Catalog # Description Current Lot Previous Lot(Valid Use Date) CAS # NDC # Unit Price Special Restriction DEC-2017) 1012288 Carotenes (3 x 100 mg) F059U0 N/A N/A 270.00 1012495 Beta Alanine (100 mg) R033Y0 F0F111 (31-AUG-2016) 107-95-9 N/A 275.00 1012509 L-Alanine (200 mg) R06030 .
1161 Fat in Meat - Soxhlet (Modified from AOAC 960.39) 1168 Moisture in Foods - Oven (Modified from AOAC 926.08,931.04, 950.46B, 925.30, 927.05, 934.06) 1190 Dietary Fibre - Insoluble and Soluble (Modified from AOAC 991.43) 1208 Sugars in Foods by HPLC (Modified from AOAC 982.14, 980.13) 1812 Fat in Milk - Modified Mojonnier (Modified from AOAC
SGPT (ALT) (Alanine amino transferase) Modified IFCC (WITH P5P) 5.5 35 CLINICAL BIOCHEM ISTRY Serum /LI- Heparin Plasma Apolipoprotein A-1 PEG immunoturbidimetric 3.9 36 CLINICAL BIOCHEM ISTRY Serum /Li- Heparin Plasma Electrolyte Chloride Indirect Potentiometry 1.5 This is annexure to 'C
Regarding the ability of HMGB1 to specifically bind bent DNA, the individual mutations of either K2 or K81 as well as the double mutation of both residues to alanine were found to completely abolish binding of truncated tail-less HMGB1 to cisplatin-modified DNA. We conclude that unlike the case with the bending ability of truncated HMGB1, where
2.1 Anatomi Telinga 2.1.1 Telinga Luar Telinga luar terdiri dari daun telinga dan kanalis auditorius eksternus. Daun telinga tersusun dari kulit dan tulang rawan elastin. Kanalis auditorius externus berbentuk huruf s, dengan tulang rawan pada sepertiga bagian luar dan tulang pada dua pertiga bagian dalam. Pada sepertiga bagian luar kanalis auditorius terdapat folikel rambut, kelenjar sebasea .
