Circuit quantum electrodynamics has proven to be a powerful tool to probe mesoscopic effects in hybrid systems and is used in several quantum computing (QC) proposals that require a transmon qubit able to operate in strong magnetic fields. To address this we integrate monolayer graphene Josephson junctions into microwave frequency superconducting circuits to create graphene based transmons. Using dispersive microwave spectroscopy we resolve graphene’s characteristic band dispersion and observe coherent electronic interference effects confirming the ballistic nature of our graphene Josephson junctions. We show that the monoatomic thickness of graphene renders the device insensitive to an applied magnetic field, allowing us to perform energy level spectroscopy of the circuit in a parallel magnetic field of 1 T, an order of magnitude higher than previous studies. These results establish graphene based superconducting circuits as a promising platform for QC and the study of mesoscopic quantum effects that appear in strong magnetic fields. A transmon qubit insensitive to magnetic fields is a crucial element in topological quantum computing. Here, Kroll et al. create graphene transmons by integrating monolayer graphene Josephson junctions into microwave frequency superconducting circuits, allowing them to operate in a parallel magnetic field of 1 T. A transmon qubit consists of a Josephson junction (JJ) shunted by a large capacitance, coupled to a high quality factor superconducting resonator. In conventional transmon devices, the resonator is fabricated from Al and the JJ is fabricated from an Al/AlOx/Al tunnel junction1, both of which cease operation above the critical magnetic field of bulk Al, ~10 mT. Even when considering alternative type II superconductors such as NbTiN or MoRe that can sustain superconductivity beyond B = 8 T7, when subjected to a strong magnetic field the superconductor will experience detrimental effects such as reduction of the superconducting gap, increased quasiparticle generation8 and the formation of Abrikosov vortices that cause resistive losses in a microwave field. In addition to disrupting the superconductivity, magnetic flux penetrating the JJ produces electron interference effects that reduce the Josephson energy EJ and strongly suppress the transmon energy spectrum. If the transmon is to be used for fast quantum gates, fast charge-parity detection and long range quantum state transfer in QC schemes10 we are compelled to consider alternatives to conventional Al based JJs. Proximitised semiconducting nanowires, acting as gate-tuneable superconductor-normal-superconductor JJs11 have been used successfully in a variety of microwave frequency superconducting circuits, allowing for studies of Andreev bound states13, electrically tuneable transmon qubits15 and transmons that exhibit substantial field compatibility16. Graphene JJs are an attractive alternative as they exhibit ballistic transport, high critical currents18 and the atomic thickness of the graphene junction greatly reduces flux penetration, protecting the JJ from orbital interference effects that would suppress EJ in high parallel fields. When combined with geometric techniques that protect the superconducting film, such as critical field enhancement19 and lithographically defined vortex pinning sites21, the transmon circuit can be protected at magnetic fields relevant to these proposals, which approach and in some cases exceed 1 T24.A superconducting transmon qubitB|| = 0 T to resolve a linewidth of ≃ 400 MHz. Although the large linewidths prevent coherent qubit control, we demonstrate the device is insensitive to the applied magnetic field up to B|| = 1 T.In this work we report the integration of ballistic graphene JJs into microwave frequency superconducting circuits to create graphene based transmons. Using dispersive microwave spectroscopy we resolve the characteristic band dispersion of graphene, and observe coherent electronic interference effects that confirm the ballistic nature of our graphene JJs. We perform energy level spectroscopy at λ/4 coplanar waveguide (CPW) resonators multiplexed to a common feedline. Each resonator is capacitively coupled to a graphene transmon, with the graphene JJ being shunted by capacitor plates that provide a charging energy EC ≃ 360 MHz. The resonators and capacitor plates are fabricated from 20 nm NbTiN due to its enhanced critical magnetic field19, and we pattern the resonators with a lattice of artificial pinning sites to protect the resonator from resistive losses due to Abrikosov vortices21. The van der Waals pickup method is used to encapsulate monolayer graphene (G) between two hexagonal boron nitride (hBN) flakes and deposit it between the pre-fabricated capacitors plates  gives a large EJ, placing ft above the resonator and giving χ a small negative value  fr Fig. . This be1 T Fig.  and calc1 T Fig.  again re1. The readout tone is set to fr whilst a second tone fd is used to drive the circuit. Excitation of the system results in a state dependent shift of the resonator frequency, and is detected by measuring the change in the complex transmission S21 at fr. At VG = 0 V (p-regime), two-tone spectroscopy at B|| = 0 and 1 T  that can be fitted to extract ft and γ, giving a minimum linewidth of 166 MHz , then deposited between pre-fabricated capacitors using a PMMA based van der Waals pickup method. Contact to the graphene stack is made by etching in a CHF3/O2 environment, followed by sputtering MoRe (t = 80 nm). As shown in Supplementary Fig. 3/O2 plasma to be 1000 × 300 nm2 in size. Device B was contacted to provide a junction length of 500 nm. The long thin leads were geometrically restricted in two dimensions, making it less favourable for vortices to form, protecting the superconductivity of the contacts proximitising the junction. The junction is then shaped in a CHF3/O2 plasma to be 500 × 500 nm2. A SiNx/Ti/Au top gate stack is then sputtered to give full junction coverage, giving greater control of μ in the junction.To fabricate the two devices (A and B) 20 nm of NbTiN is sputtered onto intrinsic Si wafers in an Ar/N atmosphere. The resonators, feedline and transmon are reactive ion etched in an SFπ and copper powder filters. The line used to drive the feedline input was heavily attenuated to reduce noise and thermal excitation of the cavity, allowing the single photon cavity occupancy to be reached. The output line of the feedline was connected to an isolator (Quinstar QCI-080090XM00) and circulator (Quinstar QCY-060400CM00) in series to shield the sample from thermal radiation from the HEMT amplifier (Low Noise Factory LNF-LNC4-8_C) on the 4 K stage. Resonator spectroscopy of device A was performed using circuit (a) to measure the amplitude and phase response of the complex transmission S21 as the frequency was varied. Resonator and two-tone spectroscopy of device B was performed using circuit (b), with a splitter used to combine the readout and excitation tones. This allows the complex S21 to be measured, but only at fixed resonator readout frequency otherwise only |S21| can be recorded.All measurements were performed in a dilution refrigerator with a base temperature of 15 mK. The samples were enclosed in a light tight copper box, and thermally anchored to the mixing chamber. An external magnetic field is applied to the sample using a 3-axis vector magnet. The two different measurement configurations used in this manuscript are depicted in Supplementary Fig. Supplementary InformationPeer Review File


O-methylation of U1 and U2 snRNAs, including at 70A in U1 snRNA and, 19G, 25G, 47U and 61C in U2 snRNA. Collectively, we suggest that TDP-43 and WDR79 have separate roles in determining CB localization of subsets of C/D and H/ACA scaRNAs.TDP-43 regulates cellular levels of Cajal bodies (CBs) that provide platforms for the assembly and RNA modifications of small nuclear ribonucleoproteins (snRNPs) involved in pre-mRNA splicing. Alterations in these snRNPs may be linked to pathogenesis of amyotrophic lateral sclerosis. However, specific roles for TDP-43 in CBs remain unknown. Here, we demonstrate that TDP-43 regulates the CB localization of four UG-rich motif-bearing C/D-box-containing small Cajal body-specific RNAs  through the direct binding to these scaRNAs. TDP-43 enhances binding of a CB-localizing protein, WD40-repeat protein 79 (WDR79), to a subpopulation of scaRNA2 and scaRNA28; the remaining population of the four C/D scaRNAs was localized to CB-like structures even with WDR79 depletion. Depletion of TDP-43, in contrast, shifted the localization of these C/D scaRNAs, mainly into the nucleolus, as well as destabilizing scaRNA2, and reduced the site-specific 2′- In nonRNP A2) ,13–17.O-methylation of RNA components of snRNPs in CBs, which lead to the last step of the assembly of snRNPs with specific RNP proteins   ,33. The d Nop58) . This CAd Nop58) ,36. In cd Nop58) ,33,35, sd Nop58) ,33,36. IAntibodies used in this study are listed in 2 in air.HeLa, 293T (HEK293 cells transformed with large T antigen) and Flp-In T-REx 293 (T-REx 293) cells (Thermo Fisher Scientific) were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum (Biowest LLC) and streptomycin  and penicillin G  at 37°C under 5% COThe construction of the plasmids expressing DAP -TDP-43  and TDP-43 deletion mutants  was described previously . To consT-REx 293 cell lines inducibly expressing protein with doxycycline were established as described previously ,39. To eg for 10 min at 4°C, the supernatant was collected as the total cell extract. For the first step pull-down, the extract (10 mg) was incubated with 30 μl of Ni-NTA Agarose (Qiagen) by rotation for 1 h at 4°C. The DAP-TDP-43 bound Agarose was washed five times with wash buffer  and eluted with 100 μl of imidazole-containing buffer  for 10 min twice on ice. The eluates recovered as His6-tagged protein complexes were diluted with 400 μl of wash buffer containing 1 mM PMSF and incubated with 15 μl FLAG M2 Agarose beads (Sigma-Aldrich) for 4 h at 4°C for the second step pull-down. After the beads were washed five times with wash buffer, the DAP-TDP-43 complexes were eluted with 150 μl of Protein-RNA extraction buffer ; 2% 2-mercaptoethanol) for 5 min at 25°C as FLAG-tagged complexes. The purified DAP-TDP-43 complexes were subjected to phenol–chloroform extraction; namely, 150 μl of eluate was added to 100 μl of phenol–chloroform (pH 8.0) (Thermo Fisher Scientific) and 15 μl of 3 M sodium acetate (pH 5.2). After vigorous mixing for 10 min at room temperature, the organic phase containing protein components and the aqueous phase containing RNA components were separated by centrifugation at 20 000 g for 10 min at 4°C. The separately precipitated proteins and RNAs by isopropanol precipitation were washed with 75% ethanol and dried. Proteins were analyzed by immunoblotting and RNAs were suspended in 100% formamide and separated on a 6% (19:1 acrylamide/bis-acrylamide) denaturing urea polyacrylamide gel with 0.5× TBE buffer. The gel was stained with SYBR Gold (Thermo Fisher Scientific) and visualized by LAS-4000 .DAP-TDP-43-expressing T-REx 293 cells (induced with 100 ng/ml of doxycycline for 24 h) were harvested, washed twice with ice-cold PBS, suspended in extraction buffer ; 1 mM phenylmethylsulfonyl fluoride (PMSF)) with 1 mM Ribonucleoside–Vanadyl Complex (New England BioLabs) and lysed by sonication using a Bioruptor 200 . After centrifugation at 20 000 http://ariadne.riken.jp/) with the human genome database (GRCh37.p5) or a human small RNA database containing all sequences of the transcripts registered in RefSeq  plus those of 18S rRNA (NR_145820) and 28S rRNA (NR_145822). The following default search parameters for Ariadne were used: maximum number of missed cleavages, 1; variable modification parameters, one methylation per RNA fragment for any residue; RNA mass tolerance, ±5 ppm, and MS/MS tolerance, ±20 ppm.In-gel RNA digestion and LC-MS analysis for RNA identification were performed as described . Assignmg for 10 min at 4°C, the supernatant was rotate-incubated with 15 μl of FLAG M2 Agarose beads (Sigma-Aldrich) for 2 h at 4°C. The Agarose beads were washed five times with wash buffer  and eluted for recovery of FLAG-tagged protein complexes with 150 μl of Protein-RNA extraction buffer  for 5 min at 25°C. Protein or RNA components were extracted as described in above section ‘Pull down of TDP-43-binding RNA for LC-MS analysis’, and subjected to the immunoblot or northern blot analysis as described below.T-REx 293 cells expressing a FLAG-tagged protein inducibly were harvested, washed twice with ice cold PBS, suspended with extraction buffer , 1 mM Ribonucleoside–Vanadyl Complex (New England BioLabs), 1 mM PMSF) and lysed by sonication using Bioruptor 200 . After centrifugation at 20 000 Immunoblot analysis was performed as described . ProteinNorthern blot analysis was performed as described . The bio2 for cross-linking and harvested with ice-cold PBS. After suspended in 1 ml of extraction buffer and 175 μl of 2.5 M NaCl  on ice for 10 min, the harvested cells were centrifuged at 20 000 g for 10 min at 4°C and used for subsequent immunoprecipitation. To isolate DAP-TDP-43 complexes, 15 μl of FLAG M2 Agarose beads were incubated for 3 h with rotation at 4°C, washed five times with wash buffer  and eluted with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer after incubation for 3 min at 65°C to recover DAP-TDP-43 complexes. The proteins were separated by SDS-PAGE on a 7% acrylamide gel and electrophoretically transferred to a nitrocellulose membrane . RNAs cross-linked with DAP-TDP-43 were detected using streptavidin-HRP and visualized by LAS-4000. RNAs sifted by cross-linking with TDP-43 were excised from the membrane, treated with 2 mg/ml proteinase K  in PK buffer  for 30 min at 37°C, mixed with 250 μl of Protein-RNA extraction buffer, and extracted with phenol–chloroform and then precipitated with isopropanol. RNAs were separated using denaturing urea PAGE with a 6% acrylamide gel and subjected to northern blot analysis.DAP-TDP-43-expressed T-REx 293 cells were cultured in a 150-mm Petri dish, exposed to UV light at 400 mJ/cmin vitro transcription of biotin-labeled scaRNAs (0.06 pmol/reaction) in the presence of 7.5 mM ATP, 7.5 mM CTP, 7.5 mM UTP, 7.5 mM GTP and 0.5 mM biotin-UTP (Roche) using CUGA7 in vitro transcription kit . The transcripts were collected by isopropanol precipitation, separated by denaturing urea PAGE on a 6% acrylamide gel and visualized by SYBR Gold staining. Each of the biotin-labeled scaRNAs was excised from its corresponding gel band, extracted from the gel and subjected to electrophoresis mobility sift assay. Primer sequences used for PCR are listed in To synthesize biotin-labeled scaRNAs, using KOD–plus-neo DNA polymerase , we first amplified DNA fragments containing a T7 promoter sequence at their 5′ ends and scaRNA2, scaRNA7, scaRNA9 or scaRNA28 at 3′ side from their corresponding template DNAs; scaRNA2-pcDNA3.1 , pCMV-gl2, 0.01% IGEPAL CA-630 (octylphenoxy poly(ethyleneoxy)ethanol), 1 mM dithiothreitol (DTT), 10 μg of yeast tRNA (Thermo Fisher Scientific), 10 μg bovine serum albumin (BSA), 10 ng of synthetic RNA, 200 ng of recombinant protein) at 25°C for 20 min. RNA–protein complexes were separated by 6% non-denaturing polyacrylamide gel electrophoresis at 100 V for 70 min in 0.5× Tris borate/EDTA buffer (44.5 mM Tris-borate and 1 mM EDTA), and transferred to a Hybond N+ membrane. The membrane was sequentially dried and UV-crosslinked using the CX-2000 UV crosslinker (UVP) at 120 mJ/cm2. The biotin-labeled scaRNAs were detected with Stabilized Streptavidine-HRP-conjugate and visualized by LAS-4000.Recombinant proteins (TF-FL and TF-TDP-43-FL) were purified and used for EMSA as described previously with some modifications . BrieflyGAPDH mRNA. Primer sequences used for qPCR are listed in Total RNA (200–300 ng) was isolated using TRIzol Reagent  and subjected to reverse transcription with random hexamer and oligo dT primers using PrimeScript RT Reagent kit . qPCR was performed using GeneAce SYBR qPCR Mix  and Thermal Cycler Dice Real Time System . The value of gene expression was calculated by delta-delta Ct method and normalized for that of Stealth RNA interference (RNAi) siRNA or Silencer Select siRNA (siRNA) and negative control siRNA or non-specific control RNA (ncRNA) were purchased from Thermo Fisher Scientific. Transfection of siRNA or ncRNA was performed using Lipofectamine RNAiMax (Thermo Fisher Scientific). In six-well plate, 100 pmol of Stealth RNAi siRNA/ncRNA or 25 pmol of Silencer Select siRNA/ncRNA was transfected into 293T or T-REx 293 cells, and 50 pmol of Stealth RNAi siRNA/ncRNA or 12.5 pmol of Silencer Select siRNA/ncRNA into HeLa cells. All siRNA sequences are listed in Total RNA (20–30 μg) was isolated from HeLa or 293T cells cultured in a 90-mm Petri dish by using TRIzol Reagent. Small RNA fraction  and large RNA fraction (containing 28S and 18S rRNA) were prepared from 10 μg of total RNA using mirVana miRNA Isolation Kit (Thermo Fisher Scientific AM1560). 28S or 18S rRNA was purified from large RNA fraction by reverse-phase LC as described in . Small Rin vitro transcription of human 13C-labeled U1 snRNA, a cDNA fragment encoding U1 snRNA was amplified by PCR from random primed HeLa cDNA library and inserted into the EcoRI/XhoI sites of plasmid pBluescript II KS(+) . 13C-labeled U1 snRNA was transcribed in vitro from SpeI-linearized plasmid (1 μg) using Megascript T3 kit (Thermo Fisher Scientific). To transcribe 13C-labeled U2 snRNA, a cDNA fragment encoding human U2 snRNA was amplified from random primed TK6 cDNA library by PCR, inserted into HindIII/XhoI sites of pcDNA3.1(+) vector using a GeneArt Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific) and used as a template DNA for the transcription after linearized with XhoI (1 μg). For RNase T1 digestion, RNA was synthesized using Guanosine-13C10 5′-triphosphate, whereas for RNase A digestion, cytidine-13C9 5′-triphosphate and uridine-13C9 5′-triphosphate were used instead of the respective 5′-triphosphate reagent that contained carbons with natural isotope distribution. The 13C-labeled U1 or U2 snRNA was precipitated in ethanol, solubilized in nuclease-free water and then purified further by reversed-phase LC as described above. The 13C-labeled 28S, 18S and 5.8S rRNA were synthesized as described  glycerol and 1 mM DTT. The guide DNA oligonucleotide was designed to hybrid with a region at the 5′ side of 17U in U2 snRNA. Prior to RNase H digestion, the LC-purified U2 snRNA was denatured at 74°C for 3 min, and annealed with guide DNA oligonucleotide by gradually lowering temperature from 74 to 50°C for 12 min. The RNase H digest was directly loaded onto reversed-phase LC columns and purified RNA fragments corresponding to the regions of 1–16 and 17–188 in U2 snRNA. Sequence of the guide DNA oligonucleotide is listed in To identify all post-transcriptional modifications, LC-purified U2 snRNA (1 pmol) was cleaved in a sequence specific manner with 3 U of RNase H  at 42°C for 30 min using a guide DNA oligonucleotide (100 pmol) in 20 μl of 40 mM Tris–HCl (pH 7.7), 4 mM MgClet al. with some modifications  were acquired at a mass resolution of 35 000. Higher-energy CID with normalized collision energy of 20 and 50% fragmented precursors. Other settings were as described  was done in 100 mM triethylammonium acetate buffer (pH 7.0) at 37°C for 60 min. RNase A digestion (0.25 ng/μl) was done with the same procedure with for RNase T1 except using internal standard RNAs synthesized with 13C-labeled cytidine and uridine. SILNAS-based quantization of 5.8S, 18S or 28S rRNA were performed as described  Triton X-100 (SIGMA) for 5 min at 25°C and then were washed again with PBST. The cells were blocked with 3% (w/v) non-fat dried skim milk in PBST for 1 h and then were incubated with an appropriate primary antibody for 2 h at 25°C. After three 10-min washes with PBST, the cells were incubated with a fluorescence-conjugated secondary antibody for 1 h at 25°C. Finally, after three additional 10-min washes with PBST, the cells were mounted using VECTASHIELD Mounting Medium with DAPI and examined with an Axiovert 200 M microscope.2, 1 mM PMSF) and lysed by sonication using the Bioruptor 200 . One-fifth of the solution was used to extract total RNA, and the remaining solution was used to prepare nucleoli. The latter solution was overlaid on 700 μl of Sol II  and centrifuged at 1700 g for 5 min at 4°C, and the supernatant was collected as the cytoplasmic-nucleoplasm fraction and subjected to the following RNA extraction. The remaining pellet was washed once with 700 μl of Sol I and centrifuged at 1700 g for 5 min at 4°C; the nucleolar fraction was collected by carefully removing the supernatant, which was subjected to the following RNA extraction. RNA extraction of each fraction was performed with TRIzol reagent. Total RNA was fixed at 4 μg, and equivalent amount of RNA from cytoplasmic-nucleoplasm or nucleolar fraction was used for northern blot analysis.Fractionation of nucleoli was carried out as described  with som6 oligonucleotide as an RNA antagonist and demonstrated that (UG)6 oligonucleotide interrupted the binding of those scaRNAs to TDP-43 , two scaRNAs—scaRNA7 (U90) and scaRNA9—are predicted to have UG-rich motifs  29,32,3,48,49. Is Figure  and F. As Figure , we suggTo locate the region bound to the C/D scaRNAs in the TDP-43 molecule, in addition to the DAP-TDP-43-expressing cell line, we established T-REx 293 cell lines that inducibly expressed each of four domain-deletion mutants having nuclear localization signals  Figure . We pullO-methylation of snRNAs -MS-based method, SILNAS  from the heavy isotope-labeled reference U1 snRNA. In fact, we detected the MS spectra corresponding to those three fragments from U1 snRNA prepared from HeLa cells treated with the non-specific control RNA (ncRNA)  Figure , left; tparation . Both thparation , and thoparation  were conparation . In contration 1;. Coincided at t3 , which aol Table . The kno0A Table , suggestO-methylation at 19G, 25G, 47U and 61C in U2 snRNA in HeLa and 293T cells, whereas it did not affect the other analyzed 2′-O-methylation sites of U2 snRNA  in the 2′-O-methylation at 40C in U2 snRNA in HeLa cells  analyses of cells that ectopically expressed one of the four C/D scaRNAs. Knockdown of TDP-43 in these cells led to re-localization of all four C/D scaRNAs from the CBs mainly to the nucleolus  and believed to cause ‘loss of function’ of TDP-43 via sequestering from its normal localization sites. Therefore, we expected that TDP-43-aggregation and the following TDP-43 loss of function might cause the de-localization of the C/D scaRNAs in CBs observed by the depletion of TDP-43. To examine this, we used a cell model causing TDP-43 aggregation by expressing TDP-12xQ/N that has tandem repeats of the prion-like Q/N-rich region of TDP-43 fused to additional TDP-43 protein sequences to trigger the formation of the aggregate of endogenous TDP-43 . In thisO-methylation at 3923G in 28S ribosomal RNA) via complementary hybridization  Figure  and H, wdization , we analhylation . In addi.8S RNAs  51,56),,56,O-met.8S RNAs .Given that WD40-repeat protein 79 (WDR79) drives the CB-specific localization of scaRNA7 and scaRNA28 (mgU2-47) via binding to the G.U/U.G wobble base-pairs in their UG-rich motifs , we deteWe, therefore, examined further the possibility that TDP-43 and WDR79 contributions to CB localization differ between C/D scaRNAs and H/ACA scaRNAs. Indeed, FISH analyses showed that WDR79 knockdown changed coilin localization into the nucleolus as reported  Supplem, whereasO-methylations of U1 and U2 snRNAs through its ability to maintain CB localization of the UG-rich motif-bearing C/D scaRNAs scaRNA2, scaRNA7, scaRNA9 and scaRNA28  mouse or those from coilin-KO Drosophila ,24,42. Icis-elements of introns has been well characterized . In. Incis-ee Figure , which re Figure . Given t-complex , our pren Figure . Therefo2+-dependent dynamic equilibrium; one has a four-helix structure and the other has a three-helix structure  via dissociation of U1 and U4 snRNPs Figure . During e Figure   complex has shown that the base at 70A of U1 snRNA stabilizes the complex via its binding to Thr89 and Ser91 of U1A protein  that are predicted to guide or inhibit post-transcriptional modifications in ribosomal RNAs ,56 Supp, TDP-43 O-methylation may be found in U1 and U2 snRNAs in individuals with ALS and SMA. In general, post-transcriptional modifications, including pseudouridylations and 2′-O-methylations in U snRNAs seem to be key regulators of pre-mRNA splicing, and thus of gene expression  ,19, de-lpression . Pre-mRNpression . Site-sppression ,49. TDP-pression . CurrentSupplementary DataClick here for additional data file.


Drug resistance of paclitaxel (TAX), the first-line chemotherapy drug for breast cancer, was reported to develop in 90% of patients with breast cancer, especially metastatic breast cancer. Investigating the mechanism of TAX resistance of breast cancer cells and developing the strategy improving its therapeutic efficiency are crucial to breast cancer cure.We here report an elegant nanoparticle (NP)-based technique that realizes efficient breast cancer treatment of TAX. Using lentiviral vector-mediated gene knockdown, we first demonstrated that TAX therapeutic efficiency was closely correlated with metadherin (MTDH) gene expression in breast cancer cell lines. This finding was also supported by efficacy of TAX treatment in breast cancer patients from our clinical studies. Specifically, TAX treatment became more effective when MTDH expression was decreased in MCF-7 cancer cells by the blocking nuclear factor-kappa B (NF-κB) pathway. Based on these findings, we subsequently synthesized a polymeric NP that could co-deliver MTDH-small interfering RNA (MTDH–siRNA) and TAX into the breast cancer tumors in tumor-bearing mice. The NPs were composed of a cationic copolymer, which wrapped TAX in the inside and adsorbed the negatively charged siRNA on their surface with high drug-loading efficiency and good stability.NP-based co-delivery approach can effectively knock down the MTDH gene both in vitro and in vivo, which dramatically inhibits breast tumor growth, achieving effective TAX chemotherapy treatment without overt side effects. This study provides a potential therapeutic strategy for the treatment of a wide range of solid tumors highly expressing MTDH.The online version of this article (10.1186/s13058-018-1042-7) contains supplementary material, which is available to authorized users. The key options in breast cancer treatment so far include surgery, chemotherapy, radiotherapy, and molecular targeted therapy . Out of Metadherin (MTDH), also known as LYRIC, AEG-1, or 3D3, has been expressed in multiple tumor types as an oncogene and associated with aberrant proliferation and drug resistance of tumor cells –10. In pWe first explored the role of MTDH gene in the TAX therapeutic efficiency toward breast cancer cells and then investigated whether a nanoparticle (NP)-based co-delivery method can be used to address TAX resistance issue in breast cancer treatment. We hypothesized that the two drugs-loaded NPs displayed onsite gene silencing in tumor tissues, which improved drug sensitivity of TAX with good tolerability. In addition, poly(lactic-co-glycolic acid) (PLGA) polymeric molecule is approved by the US Food and Drug Administration with high biosafety; this study thus presents a promising strategy for clinical practice.This is a retrospective study that was approved by the Fourth Hospital of Hebei Medical University in Shijiazhuang, China. In this study, we selected 44 cases with breast cancer, which were proven to be breast invasive ductal carcinoma by pathological diagnosis from March to December 2010. The median follow-up time was 84 months (range of 8–90 months). The primary endpoints were disease-free survival (DFS) and overall survival (OS). We detected the samples from carcinoma of 44 patients before neoadjuvant chemotherapy. Immunohistochemistry was used to detect MTDH expression in all tissues. All the patients were recommended to use combination chemotherapy of taxol and anthracycline. Clinical benefit was evaluated by the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) and pathologic complete response (pCR) rates after neoadjuvant therapy. pCR was defined as ypT0 ypN0. All of above procedures were approved by the Fourth Hospital Ethics Committee of Hebei Medical University in Shijiazhuang, China (SCXK2009–0037).All of the immunohistochemistry slides for MTDH were reviewed again by two independent pathologists. Immunohistochemistry staining of 4-μm sections of formalin-fixed paraffin-embedded tissue was rehydrated and incubated with anti-MTDH primary monoclonal antibodies  or phosphate-buffered saline (PBS) at 4 °C overnight, followed by sequential incubation with MaxVision™/horseradish peroxidase (HRP) and diaminobenzidine (DAB). Then slides were counterstained with hematoxylin, dehydrated, and mounted.The levels of MTDH expression were evaluated on the basis of the staining intensity (SI) and percentage of positively stained tumor cells (PP). SI was defined as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). PP was graded according to the following criteria: 0 (no positive tumor cells), 1 (1%–20% positive tumor cells), 2 (21%–50% positive tumor cells), 3 (51%–70% positive tumor cells), and 4 (>70% positive tumor cells). The immunoreactive score (IRS) was calculated as follows: IRS = SI × PP. IRS of 0 means negative expression, IRS of 1–3 means weakly positive, IRS of 4–6 means moderately positive, and IRS of 8–12 means strong positive. Here, low expression was defined as an IRS of 3 or less. High expression was defined as an IRS of 4 and more .2 at 37 °C. TAX was purchased from Yangzijiang Medicine Co. Ltd. .The human breast cancer cell lines MCF-7 and MDA-MB-435S were purchased from the American Type Culture Collection  and propagated in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and antibiotics. All cells were maintained in 5% COEscherichia coli. DH5α. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of MTDH mRNA and protein of MTDH-shRNA to verify the effect of transfection. Plasmids enveloped in lentivirus were incubated with MCF-7 cells for 6 h according to the MOI (multiplicity of infection) value and the virus titer and subsequently placed in fresh medium. Puromycin (0.4 μg/mL) was used to screen stable transfection cell lines.The plasmids containing MTDH gene or MTDH-short hairpin RNA (MTDH-shRNA) (5′-gcaattgggtagacgaagaaa-3′) were designed and amplified by transfecting into −∆∆Ct. The data were normalized to the geometric mean of housekeeping gene β-actin to control the variability in expression levels. RT-PCR primers were synthesized by SBS Genentech Co. Ltd. . The specific primers for MTDH and reference gene (β-actin) are as follows:MTDH forward: 5′-AAATAGCCAGCCTATCAAGACTC-3′;MTDH reverse: 5′-TTCAGACTTGGTCTGTGAAGGAG-3′.β-actin forward, 5′-GCTACAGCTTCACCACCACAG-3′;β-actin reverse, 5′-GGTCTTTACGGATGTCAACGTC-3′.Total RNA from treated cells was extracted with Trizol  in accordance with the instructions of the manufacturer. Total RNA was used to synthesize cDNA by using PrimeScript RT reagent Kit (Takara). Then RT-PCR was carried out using Power Up SYBR Green Master Mix . The reaction was conducted using the following parameters: 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 30 s during 40 cycles. Internal control and primers for RT-PCR were obtained from the reference. RT-PCR was then employed to determine the change of MTDH mRNA in MCF-7–MTDH cell line and MCF-7–MTDH–shRNA cell line. The experiments were repeated for three times and data were analyzed using 2Cells were lysed and total proteins were separated by 10% SDS-PAGE and transferred  onto a PVDF membrane. After blotting with 5% nonfat milk, the membranes were incubated with primary antibodies  1:1000, anti-IκBα1:1000, and β-actin 1:1000) at 4 °C overnight. Then the membranes were washed by TBS-T buffer and incubated with secondary HRP-labeled anti-rabbit antibody at room temperature for 1 h and washed with TBS-T buffer three times (10 min each time). The target proteins were visualized with a chemiluminescence system  and normalized to β-actin from the same membrane.Cell apoptosis was performed using an Annexin V-PE/7-AAD Apoptosis Detection Kit . The experiments were carried out strictly in accordance with the instructions of the manufacturer. The cells were then analyzed by Beckman Coulter Cytomics FC 500 flow cytometry . The data were analyzed by EXPO32 ADC analysis software. Cell cycle analysis was performed by using the standard method with some modifications. In brief, cells were fixed with 75% ethanol at 4 °C overnight. The fixed cells were washed by PBS and suspended with 200 μL RNaseA at 37 °C for 10 min, and 250 μL PI (100 μg/mL) was added to stain the DNA of cells in the dark for 15 min. Cell cycle was analyzed with a Beckman Coulter Cytomics FC 500 flow cytometry, and the data were analyzed by Multicycle AV for Windows (version 295) software.4/well (TAX 0 μg/mL) or 5 × 104/well (TAX 1 μg/mL) in 100 μL RPMI-1640 medium. After incubation in 5% CO2 at 37 °C overnight, the RPMI-1640 medium in each well was replaced with a different concentration of TAX (0 and 1 μg/mL) and further incubated for 0, 24, 48, and 72 h. Afterwards, 10 μL of CCK-8 was added to each well for another 2 h at 37 °C. The absorbance at 450 nm was read by the Microliate Reader . The inhibitory rate of cell growth was calculated on the basis of the following equation: cell growth inhibition rate =  × 100%. The experiments were repeated three times.Cell viability was determined by a Cell Counting Kit-8 (CCK-8) assay. MCF-7, MCF-7-vector, MCF-7–MTDH, and MCF-7–MTDH–shRNA cells were seeded into 96-well plates at a density of 1 × 106 in 0.1 mL) were injected subcutaneously into 4-week-old female nude mice, respectively. TAX treatment was started at the third week after cell injection. The mice were randomly assigned to an untreated group and TAX treatment groups. The dose of TAX was 10 mg/kg and administered by intraperitoneal (IP) injection once a week for a total of four injections. Tumor volume was measured every three days . After the last treatment, the mice were sacrificed and the tumors were removed for weight analysis.MCF-7, MCF-7-vector, MCF-7–MTDH, and MCF-7–MTDH–shRNA cells (5 × 10For poly(etherimide)-poly(lactic-co-glycolic acid) (PEI-PLGA) NP synthesis, 20 mg was dissolved in 1 mL of methylene chloride and mixed with 0.2 mL deionized water. The mixture was emulsified through sonication by using probe sonicator at 25% power for 5 min. Then 2 mL of 2% poly (PVA) and 0.2 mL of hydrophobic TAX (with different ratios) dissolved in methylene chloride were added into the mixture. The solution was emulsified again at 30% power for 5 min, added dropwise into 10 mL of 0.6% PVA, and stirred for 3 min. The organic solvent was removed in a rotary evaporator under reduced pressure. The core particles containing TAX were centrifuged at 12,000 revolutions per minute (rpm) for 5 min and rinsed with deionized water. For loading siRNA, different ratios of TAX were added into the solution of core NPs and stirred at a rate of 200 rpm for 20 min. The core NPs absorbed by siRNA on their surface were centrifuged at 12,000 rpm for 5 min.The NPs were negatively stained with uranyl acetate solution (2%) and deposited on a carbon-coated copper grid. The morphology was characterized with a transmission electron microscope (TEM) . Size distribution (diameter in nanometers) and surface charge  of NPs were determined by using a ZetaSizer Nano series Nano-ZS  equipped with a He-Ne Laser beam at a wavelength of 633 nm and a fixed scattering angle of 90. Determinations were performed at 25 °C for samples appropriately diluted in distilled water.6 cells in 50 mL PBS) mixed with 50 mL of Matrigel were transplanted into the mammary fat pads of the mice and allowed to grow to a tumor size of about 100 mm3 . The mice were then randomly divided into different experimental groups. All procedures were approved by the Committee on the Ethics of Animal Experiments of the Health Science Center of Peking University .Six-week-old female BALB/c nude mice were purchased from Beijing Vital River Laboratories . Human breast cancer cells  and the least significance difference (LSD) multiple comparisons test with PASW Statistics 23. Tumor volumes were compared by using a Kruskal–Wallis test followed by the Mann–Whitney test.In a cohort of 44 neoadjuvant chemotherapy breast cancer patients, 29 patients were MTDH gene positive and 15 patients were negative. Patients with high MTDH protein expression  via RT-PCR and Western blot  that allows both MTDH–siRNA and TAX to be concurrently delivered into breast cancer cells to effectively control the breast cancer condition. After emulsification twice, the TAX was loaded into the hydrophobic layer and the MTDH–siRNA was bound at the NP surface after addition through electrostatic interactions. Figure Two prerequisites for efficient siRNA-mediated gene silencing effect are high siRNA uptake levels and successful release of siRNA to cytoplasm. To study cellular uptake of NPs, we labeled NP-TAX–siRNA with near-infrared fluorescent dye Cy5. After incubation of MCF-7 cells with NP-TAX–Cy5–siRNA for 6 h, obvious red fluorescence appeared in the cytoplasm Fig. . In addi3, Cy5.5-labeled NP-TAX–siRNA were injected by the tail vein. In vivo imaging results showed that the NPs gradually accumulated in tumor sites as a function of time   Fig. . TreatmeTo confirm the role of MTDH knockdown in the interference of tumor growth, we sectioned tumors and analyzed the levels of MTDH protein. Figure 0/G1 phase [MTDH was identified as an oncogene that functions in both drug resistance and metastasis . UpregulG1 phase .0/G1 phase cells and reduction of S and G2/M phase cells but that MTDH overexpression induced cell cycle arrest in S phase. Additionally, knockdown of MTDH inhibits the growth of xenograft tumor in vivo. Taken together, our results suggest that MTDH expression plays a crucial role in the MCF-7 breast cancer cell proliferation and is potentially useful for breast cancer treatment.In our study cohort of 44 patients with breast cancer, we found that MTDH expression was negatively correlated with the probability of DFS and efficacy of TAX treatment, which should be further confirmed in a large population of patients with breast cancer. At cellular level study, we tested the cell proliferation of MCF-7–MTDH and MCF-7–MTDH–shRNA cells by CCK-8 assay and found that MTDH knockdown inhibits cell growth. The results of flow cytometry demonstrated that knockdown of MTDH resulted in an increase of G2/M phase and induces cell apoptosis [2/M phase in MCF-7–MTDH–shRNA treated with TAX was significantly higher than that in the control and MCF-7–MTDH groups. In in vivo experiments, compared with the MCF-7–MTDH and control groups, the volume of MCF-7–MTDH–shRNA xenograft tumors treated with TAX was significantly smaller. These data together suggest that overexpression of MTDH resisted TAX but that MTDH knockdown increased the sensitivity of MCF-7 cells to TAX treatment. In addition, MTDH plays a key role in the activation of diverse signaling pathways, including PI3K/Akt, NF-κB, and Wnt/β-catenin pathways [As one of the most important anticancer drugs, TAX has been widely used for chemotherapy in various malignant tumors for about 40 years  and is tpoptosis –25. Howepathways . The actpathways , 28. We Chemoresistance is currently the major cause for breast cancer treatment failure, especially for metastatic breast cancer. Numerous siRNAs have been demonstrated to be effective for in vivo tumor growth modulations , but theIn summary, we have revealed, for the first time, that overexpression of MTDH in breast cancer cells is related to TAX chemotherapeutic drug resistance. To achieve in vivo therapeutic assessment by tumor-specific knockdown of MTDH, we have devised a polymer-based nanocarrier to co-deliver anti-MTDH siRNA and TAX into tumor tissues. The designed NPs were composed of a cationic copolymer, which wrapped TAX in the inside and adsorbed the negatively charged siRNA on their surface. After systemic administration, the NPs had good tumor-targeting ability based on the EPR effect of tumor vasculatures and displayed effective antitumor activity without overt side effects. Based on our study, we provide a new strategy for reversing TAX resistance in breast cancer treatment, especially for those with high MTDH protein level.Additional file 1:Table S1. Characteristics of 44 breast cancer patients with neoadjuvant chemotherapy. Figure S1. The MTDH mRNA and protein expressions level in different groups after transfecting. We constructed four groups of shRNA and one control shRNA. Then we selected the optimal silent shRNA via real-time PCR and western blot. Figure S2. Annexin V-PE/7-AAD assay for determination of apoptosis of cells overexpressing or knocking down metadherin (MTDH) with a flow cytometer. Annexin-positive cells are presented in gate 4. Figure S3. Annexin V-PE/7-AAD assay for the determination of apoptosis of different cells after paclitaxel (TAX) treatment. The apoptosis rate of MCF-7–metadherin–short hairpin RNA (MCF-7–MTDH–shRNA) cells was significantly enhanced. Figure S4. Flow cytometry was adopted to analyze cell cycle after cells overexpressing or knocking-downing MTDH. Compared to MCF-7 cell, the MCF-7-MTDH cell had more S phase and less G0/G1 and G2/M phases, while knockdown of MTDH did the opposite. Figure S5. Cell cycle assay for different cells after paclitaxel (TAX) treatment. Compared with MCF-7 and MCF-7-vector, the G2/M phase rate of MCF-7–metadherin–short hairpin RNA (MCF-7–MTDH–shRNA) cells was significantly enhanced. While overexpression of MTDH did the opposite. Figure S6. Paclitaxel (TAX) release from the polymer nanoparticles (NPs). The NPs showed a faster release rate for TAX over time in PBS at pH 4.4 than at pH 7.4. Each bar represents the mean ± standard deviation of three replicates. Figure S7. In vivo tumor targeting of nanoparticles (NPs). Nude mice bearing MCF-7 tumors (~100 mm3) were given a single intravenous injection of Cy5.5-labeled free small interfering RNA (siRNA) or NP-TAX–siRNA by the tail vein. In vivo fluorescence signals were recorded by using a Maestro2.10.0 imaging system for up to 24 h post-injection. Abbreviation: TAX paclitaxel. (DOC 2597 kb)


For this reason, we evaluated the performance of graphene-based heaters with from one to five layers of graphene on flexible and transparent polyethylene terephthalate (PET) substrates in terms of their electrothermal properties like heating/cooling rates and steady-state temperatures as a function of the input power density. We found that the heating/cooling rates followed an exponential time dependence with a time constant of just below 6 s for monolayer heaters. From the relationship between the steady-state temperatures and the input power density, a convective heat-transfer coefficient of 60 W·m Transparent resistive heaters were proposed for a variety of applications, such as sensors , displaysp2-bonded carbon atoms in a honeycomb pattern [3, Au-CH3NO2, or HNO3 to enhance their electrothermal performance [The electrothermal properties of graphene, an atom-thick planar sheet of  pattern ,13,14, w pattern , indicat pattern ,17,18, a pattern . Among t pattern . Graphenformance . HoweverIn this article, we present the results of a study of the electrothermal properties of transparent undoped few-layer graphene-based heaters where from one to five layers of graphene grown by CVD were transferred to flexible polyethylene terephthalate (PET) substrates. Our observations of their heating/cooling rates at different power densities are reported in the upcoming sections.For the set of experiments presented in this work, monolayer graphene was grown on copper foils under low partial pressure by CVD following a standard procedure previously described in detail . In shorAfter growth, a standard layer-by-layer approach was employed to transfer the graphene from the copper foil onto PET substrates involving spin-coated poly(methyl methacrylate) (PMMA). By repeating the transfer process, a set of samples with between one and five layers of graphene was obtained. Samples were then turned into heaters by deposition of Cr/Au electrodes with a thickness of 10/70 nm at the edges of the graphene on PET samples. Finally, T-type thermocouples were attached to the back side of the PET substrates for in situ monitoring of the heater temperature by using a Keysight 34,970 A data acquisition/data logger switch unit. The accuracy of the thermocouple was estimated to be ±0.5 °C. All measurements were performed in a fume cupboard.After fabrication, the quality of the graphene was investigated. Optical images showing the morphology of the Cu foil after graphene growth are shown in 2 is shown in The flexibility and transparency of a monolayer graphene heater sample with an active heating area of ~1.5 × 1.5 cm0 is the time when the cooling starts.The heating mechanism of the graphene heaters was Joule heating. The electrothermal performance of monolayer graphene-based heaters determined using T-type thermocouples is shown in 2 area of the graphene heater. The choice of plotting versus the power density was done to enable a comparison between the performance of our graphene heater and other heaters previously reported in the literature. The plot in From these experimental plots of the heating/cooling behavior of the graphene heaters, we could also extract the steady-state temperatures versus the input heating power. As shown in h, of the graphene heaters could be determined from the trendline slopes of the data for each type of graphene heater, with a calculation method similar to a previous report [T = T − T0 is the temperature difference between the steady-state temperature, T, and the ambient temperature, T0. The resulting heat transfer coefficients are shown in h ≈ 60 W·m−2·°C−1). Similarly, the Pt thin-film heater had a heat transfer coefficient of ~150 W·m−2·°C−1, while the corresponding value for the silver paste heater was ~240 W·m−2·°C−1.From the data in s report . Theoret−2·°C−1), but in the range for that of forced convection of air (~20–200 W·m−2·°C−1). The higher value obtained here may be due to our experiments being conducted in a fume cupboard. Deviations between graphene samples were not appreciable, thereby validating the reliability of our measurements. The differences in heat-transfer coefficients between carbon-based heaters and metal thin-film heaters may be attributed to differences in the thermal interface conductance between the solid-gas adsorbates.The heat-transfer coefficients obtained for the graphene-based heaters were higher than the coefficient for natural convection of air (~5–25 W·mFinite element models were developed in COMSOL for studying the steady-state properties of graphene-based heaters and their surface temperature distributions. The electrically generated heat was modeled by using the electric currents and layered shell interface aimed at computing currents and potential distributions in thin conducting layers. Simulations using COMSOL for modeling properties of graphene were reported in previous work . In thisFinally, an interesting observation made during our experiments was that the resistance of the graphene-based heaters increased after some time of exposure to air. We found that the resistance of the transferred graphene films was significantly increased after a one-year exposure to air, with increases for some samples as much as three to five times, which is much larger than reported in a previous study . Other sBased on the assumption that graphene, with its excellent electrothermal properties, could be a perfect material for transparent heaters in many applications, we designed and fabricated a set of graphene-based heaters with from one to five layers of CVD graphene grown on copper and transferred to a PET substrate. The properties of these graphene-based heaters were evaluated both experimentally and theoretically by simulations in terms of steady-state temperatures and in terms of heating/cooling rates versus the applied power density. In conclusion, we find our results promising in terms of quantifiable parameters such as thermal time constants, maximum heating/cooling rates, and convective heat-transfer coefficients when compared to Ag and Pt metal-based thin-film heaters. However, much work remains to refine the fabrication process and to improve the quality of the graphene films. As an example, we found that the quality of the graphene film and its adhesion to the PET substrate play a key role in determining the performance and reliability of graphene-based heaters when it comes to their electrothermal properties. The results presented here in this study strengthen our belief in graphene-based heaters as being promising candidates for the next generation of transparent heaters for various applications, such as anti-fog windows, mirror defoggers, and outdoor displays.


Square millimeters of free-standing graphene do not exist per se because of thermal fluctuations in two-dimensional crystals and their tendency to collapse during the detachment from the substrate. Here we form millimeter-scale freely suspended graphene by injecting an air bubble underneath a graphene monolayer floating at the water–air interface, which allowed us to measure the contact angle on fully free-standing non-contaminated graphene. A captive bubble measurement shows that free-standing clean graphene is hydrophilic with a contact angle of 42° ± 3°. The proposed design provides a simple tool to probe and explore the wettability of two-dimensional materials in free-standing geometries and will expand our perception of two-dimensional materials technologies from microscopic to now millimeter scales. Experimental determination of the contact angle of a two-dimensional film is crucial to understand its wettability characteristics. Here, the authors use the captive bubble method to estimate a contact angle value of 42° ± 3° for a monolayer graphene film. Although graphene was long believed to be a graphite-like material7, some recent studies have shown a wide spread of water contact angle (CA) on graphene10, with values ranging from 10° when supported by water10 to 127° on solid substrates6. One reason for such discrepancies in the values of the contact angle is the difference in sample preparation and measurement conditions8. The adsorption of airborne hydrocarbons, the cleanliness and quality of the graphene–substrate and graphene–water interface can have significant effects on the measured contact angles, which, however, can be minimized in most cases by conducting experiments in controlled atmospheres and by avoiding the use of polymers during the transfer process14.The wetting properties of graphene have been a subject of intensive theoretical and experimental investigations over the last decade. Extremely thin and electrically conductive, graphene is widely used in biosensors, lab-on-a-chip and microfluidics platforms where graphene is in contact with water, vapor and analytesThe wetting characteristics of a material are dictated by both the surface and the bulk properties of the material, which implies the impossibility to determine the intrinsic wetting properties of two-dimensional (2D) materials which have no bulk. In other words, all wetting characteristics of graphene, such as contact angle and surface energy, refer not only to the graphene surface but also to the bulk phase underneath it and must not be regarded as solely graphene’s properties.16. Being the only experimental indication of free-standing graphene’s wettability up to now, the contact angle of partially suspended graphene is still an indirect measure and requires multistep sample preparation which may result in an ill-defined graphene–substrate interface yielding a range of contact angle values.In this respect, probing the wetting characteristics of free-standing graphene can give an indispensable insight for understanding graphene’s wettability. Yet, due to the extreme fragility of graphene and other 2D materials, studies on free-standing graphene have been limited to theoretical predictions with only a few experimental works on partially suspended graphene18.In this work, we present a simple and clean captive bubble design for measuring directly the wettability of free-standing graphene. The captive bubble method, i.e., the injection of an air bubble underneath graphene floating on water, allows for the formation of a graphene free-standing area as large as 1.5 by 1.5 mm, the largest free-standing area that has been reported so far for a 2D material. Essentially, graphene remains floating on the water surface after copper etching, intrinsically preventing any transfer- or handling-related contamination and corrugation. An additional advantage is that the graphene side on which the contact angle is measured  has never been exposed to ambient air and is therefore not subjected to airborne hydrocarbons adsorption20. The captive bubble and sessile drop configurations represent the same three-phase equilibrium and, therefore, are equivalent , allows for a reliable contact angle measurement. Advantages of this method in comparison with the sessile drop technique is that deionized water is primarily composed of water molecules . Another remarkable advantage of the technique is that the bubble is saturated with water, therefore yielding a contact angle in equilibrium in time.The difficulty of the contact angle measurement on free-standing graphene is that 2D materials do not withstand the mechanical disturbances originating from—for example—depositing a droplet of liquid on their surface because of their extreme thinnessFor the comparison of the captive bubble method with the sessile drop technique the water contact angle was measured on highly oriented pyrolytic graphite (HOPG). HOPG was exfoliated with the scotch tape in air or in water depending on the method of contact angle measurement. The average contact angles are 59° ± 3° for the sessile drop method and 60° ± 3° for the captive bubble method Fig. . Both me11. A surface-energy analysis using the Owens–Wendt method  (PMMA) were measured using the captive bubble method and the sessile drop technique as control tests respectively. Graphene appeared wetting transparent in both cases displaying the contact angles of the bare PMMA support—that is, 53° ± 4° measured by the captive bubble method Fig.  and 54° The agreement between the sessile drop and captive bubble results for freshly exfoliated graphite and graphene/PMMA in which graphene was not exposed to air shows that, although the air in the bubble may contain hydrocarbon contaminants, they do not affect the contact angle because of the short-lived graphene-bubble contact and/or negligible amount of hydrocarbons present in the bubble.For contact angle measurement on graphene using the captive bubble technique, an air bubble is deposited using an inverted needle underneath graphene Fig. .Fig. 2Ca28. According to Neumann’s theory the total contact angle on a deformed substrate can be described as a sum of two angles, beneath and above the contact line, i.e., θabove + θbelow   Fig. .The measurements of the contact angle of an air bubble on graphene, thus, are more complex than measuring the contact angle of a drop of water on graphene, and consist of measuring the contact angle measurement above and below the three-phase contact line. The schematic and optical images of an example of a measurement of the contact angle of water on graphene are shown in Fig.  θbelow and of the inflection angle θabove, but the difference between the two, i.e., the actual contact angle, is independent of the bubble volume and equal to 42° ± 3°: for a bubble volume of 6 µl, the resulting contact angle is 42° , and for a bubble volume of 0.2 µl, the measured angle is 42° and there is no observable inflection to account for as the smaller bubble does not induce significant stretch in the graphene sheet  did not exhibit appreciable difference in the water contact angle Fig. . Since dins Fig. .Fig. 3Gr36. In order to test the effect of the environment on one side of graphene on its hydrophobicity on the other side, we performed the experiments under different humidities. We measured contact angles of water using the captive bubble method with humidities of 98%, 85%, and 50% regulated by saturated salt solution of K2SO4 and KCl37 (see Methods for more details). Interestingly, a higher humidity level yields lower water contact angle: 29° ± 8° at 98% humidity and 42° ± 7° at 50% humidity  grown graphene is not monocrystalline and has grain boundaries, which could make graphene permeable to air39. In our experiments, the air bubble underneath graphene was stable from two seconds up to fifteen minutes after which either the bubble or graphene would collapse .As for all microscopic approaches, the captive bubble method is technically challenging for studying 2D materials because they are fragile and even small vibrations can break them apart. In some cases, cracks and holes appear on the graphene surface during the etching process16, our findings demonstrated that clean fully free-standing graphene is mildly hydrophilic . However, such low contact angle is rather surprising, because given the wetting transparency of graphene10, the contact angle of free-standing graphene should be identical to the contact angle of air, i.e., 180°. The wetting behavior, therefore, in this case cannot be only dictated by the transmission of water–air interactions, but is substantially affected by the phenomena occurring at the graphene surface. Remarkably, the measured contact angle values for mono-, bi-, four-layer graphene, and graphene treated with O2 and H2 plasma are similar  and graphene. As a complementary evidence for the hydrophilic behavior of graphene in water, stable surfactant-free dispersions of graphene have been recently obtained in degassed water44. The apparent inability of graphene to form stable aqueous dispersions, which was previously attributed to the hydrophobicity of graphene, is now explained by the adsorption and further coalescence of nanobubbles on the graphene surface.Although partly suspended graphene on a texturized substrate shows hydrophobic properties with contact angle up to 85°lar Fig. , also suOn the other hand, an increase in the environment humidity, i.e., the concentration of water molecules on the top side of graphene, leads to a decreasing water contact angle value and therefore an increase in the hydrophilicity of graphene Fig. , indicatIn conclusion, we have obtained a millimeter in size-suspended 2D material by simply harvesting surface tension forces at the air–water–graphene–air interface using an air bubble captivated on graphene floating on water.Direct contact angle measurements have shown that free-standing graphene has hydrophilic properties. Advantageously to other methods, our technique allows to probe the very water–graphene–air interface, in the cleanest way, avoiding any irregularities arising from the transfer and handling processes. The observed hydrophilicity could be explained by the formation of hydrogen bonds which would impact the spontaneous adsorption of water on the graphene surface.We believe that this work provides a stimulus to further study the still unexplored basic properties of suspended 2D materials, as their surface chemistry, surface energy, compressive and flexural strength, and device interaction at a millimeter-scale level in a free-standing geometry.45. Before conducting contact angle experiments, the backside of graphene-on-copper (G/Cu) was removed with a O2 plasma. Both types of graphene  showed the same results for water contact angle measurements.Two types of graphene were used: monolayer graphene on a copper substrate provided by Graphenea and graphene grown in a tube oven on a 25 µm copper foil at 1035° according to the procedure described in ref. 46 of graphene on G/Cu48.Multilayered graphene was prepared by repetitive PMMA transferHighly oriented pyrolytic graphite  was purchased from NT-MDT.45. In general, the presence of ions in water has a very small effect on the surface tension of water—in the order of 3% or lower at the concentration of 0.3 M51—and, therefore, negligible effects on the measured contact angle. Consequently, and given the precautions we undertook to replace the etching APS solution by water, we assume that possible presence of residual ions had no effect on the contact angle measurements  (98% Sigma-Aldrich). Once the copper foil was etched away, the APS solution was repeatedly replaced with ultrapure water by sequential diluting steps yielding a clean graphene surface without any observable APS crystalsTo place a 6 µl air bubble under the water–graphene–air interface, air was injected through a J-shaped inverted needle underneath the graphene Fig. . The consn-glycero-3-phosphocholine (DPPC) lipids (Avanti Polar Lipids Inc.) at a surface pressure of 30 mN m−1 as it is described in refs. 52. The lipids had a concentration of 1 mg mL−1 and were first dissolved in CHCl3/CH3OH 3:1 vol %. First, graphene on copper45 (copper facing down) was placed floating on the etchant solution and the appropriate amount of lipids (depending on the size of the graphene and of the cuvette) was added on the surface of the etchant solution around graphene. The etchant solution was then sequentially replaced with ultrapure water and the contact angle was measured. The lipids are known to only spread on the surface of water (around the graphene) without adsorbing on its surface (as measured by infrared spectroscopy)40.To improve the stability of graphene on the water surface, graphene was surrounded with a Langmuir–Blodgett film of 1,2-dipalmitoyl-Graphene surrounded with lipids showed a higher stability during the deposition of the air bubble. Both graphene samples, without and with lipids, showed similar measured contact angles, i.e., 42° ± 3° and 42° ± 3° respectively  plasma in a computer controlled Diener plasma generator for 247 s 34.For contact angle measurements of hydrogenated and oxygenated graphene, graphene was first hydrogenated  using a H53 at room temperature using a 100× objective and 457 nm and 532 nm lasers at a power below 2 mW to avoid excessive thermal damage of graphene. Figure 2 wafer (b). The shape of the 2D peak (~2700 cm−1), that can be fitted with a single Lorentzian component is indicative of single-layer graphene53. The absence of a D peak at ~1370 cm−1 /I(G) decreased from ~2 (pristine graphene) to ~1 after 4 min of hydrogen plasma treatment, indicating the effective doping in the lattice induced by hydrogenation54. Moreover, the appearance of a D′ peak (~1620 cm−1) in hydrogenated graphene is also related to the activation of defects. The I(D)/I(D′) value of ~10 further confirms the sp3 nature of hydrogenated defects55.For hydrogenated and oxygenated graphene, however, the appearance of the D peak Figure  results Optical images of graphene on water and graphene transferred on silicon wafer were taken with a Leica optical microscope (DM 2700 M).Contact angle measurements were conducted with a standard Ramé-Hart 250 goniometer  and recorded with the DROPimage advanced v 2.8 software under ambient conditions (22 °C). Two methods were used for the characterization of wetting. For the sessile drop technique a water droplet of 5–7 μL was deposited on a substrate and contact angle was measured within five seconds. For the captive bubble method, an air bubble with a volume of 6 μL was supplied with a microsyringe at the interface with an inverted needle . The analysis of contact angles from recorded videos were made with the software ImageJ .59. For that, an oversaturated salt solution is placed in a closed box and certain equilibrium vapor pressure (and thus relative humidity) is created. The oversaturation of the solution assures that the built vapor pressure is stable to presence of moisture sources and sinks  and, therefore, provides a precise humidity level. Different salts have different saturated vapor pressures at a given temperature, and the humidity thus can be varied by changing the chemical composition of the salt.Experiments with controlled humidities were carried out using the saturated salt solution method, commonly used for accurate humidity control and the calibration of hygrometers37 and K2SO4 for the humidity of 97.59 ± 0.53%37. For measurements at every given humidity, a beaker with the corresponding salt solution was placed in a sealed glass chamber with an embedded syringe (for further contact angle measurements) together with the cuvette containing graphene floating on water. Then the contact angle was measured using the captive bubble method. The relative humidity of 50% was the standard ambient humidity of the laboratory maintained by a moisture extractor and measured by a hygrometer, and the contact angle measurements were conducted without salt solutions.For our experiments, we used oversaturated solutions of KCl for the humidity of 85.11 ± 0.29%59. Ultrapure water, ethanol (96%), ethylene glycol , diiodomethane (99%), and methylnaphthalene (95%) were used as test liquids. Details on measured contact angles, surface tension of liquids and surface-energy calculation are provided in the Supplementary information (Supplementary Note The surface energy and its components were calculated from the contact angle measurements of different liquids on target surfaces using the Owens–Wendt techniqueSupplementary informationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


A humidity sensor using suspended carbon nanotubes (CNTs) was fabricated using a low-temperature surface micromachining process. The CNTs were functionalized with carboxylic acid groups that facilitated the interaction of water vapor with the CNTs. The humidity sensor showed a response time of 12 s and a recovery time of 47 s, along with superior hysteresis and stable performance. The hysteresis curve area of the suspended structure is 3.6, a 3.2-fold reduction in comparison to the non-suspended structure. A comparative study between suspended and non-suspended devices highlights the advantages of using a suspended architecture.  Carbon nanotubes (CNTs), first discovered by Iijima , are oneThere are many works in the literature focusing on the fabrication of humidity sensors based on CNTs. Multiwalled CNTs (MWCNTs)-based humidity sensors were fabricated and studied by many research groups e.g., ,10,11). ,11. 10,12 [Suspended CNTs have also been used previously to detect gases such as NO2 . One of 2 . This, c2 . Reduced2 , phonon 2 , etc. ThIn our previous work , the advThe detailed fabrication process has been discussed previously in . NotablyThe process is carried out on a silicon (Si) substrate. SU8, an epoxy-based negative photoresist is used as a sacrificial layer to obtain suspended CNTs. SU8 was chosen as the sacrificial material due to its uniform surface profile and ease of availability. The SU8 is partially crosslinked by UV lithography. The crosslinked SU8, of thickness 3.6 µm, is used as an anchor for the CNT beam, while the uncrosslinked SU8 is used as a sacrificial layer. The uncrosslinked SU8 is prevented from dissolving in further processing steps by depositing a 60-nm barrier layer of aluminum by filament evaporation. The CNTs used in this work are obtained pre-functionalized with a carboxylic acid group (‒COOH) from Carbon Solutions Inc.  and used without any further material processing. 0.125 g of the as-obtained CNTs in powder form were dispersed in a 1 wt % SDS solution. The as-prepared solution was then ultrasonicated for 6 h at room temperature, resulting in a uniformly dispersed CNT solution. The solution is then ultra-centrifuged at 47,300 rpm for 60 min. The top half of the solution is then decanted for further use. Then, the CNT films are formed using vacuum filtration. Vacuum filtration enables the formation of a homogenous film with a uniform distribution of CNTs. This process also ensures strict control over film thickness. The CNT films used here are 0.7 µm thick. The 20 nm thick aluminum (Al) electrodes are deposited on the CNTs before release to form the suspended CNT beams. The schematic of the resulting suspended CNT beam cross section is shown in The device characterization is done in a humidity chamber connected to an external humidifier, which acts as the humidity source. The sensors are interconnected using probes aligned using micro-positioners. The humidity percentage in the chamber was verified using a high-precision sensor embedded within the chamber. The resistance of the devices was measured using a Keithley Digital Multimeter  connected to a computer for data acquisition. To study the humidity sensing properties of the CNT-based sensors, the resistance of the devices was measured as a function of relative humidity. The humidity was measured from a base of 15% RH to a maximum of 98% RH gradually increased in steps of ∼10% RH per 2 min. The results are shown in The response time, defined as the time taken to achieve 10‒90% of the total resistance change, is measured to be 12 s for the suspended sensors and around 28 s for the non-suspended sensors, as shown in Consistent performance of the sensor is necessary for long-term applications. Thus, repeatability of the sensors was verified by subjecting them to continuous humidity cycles by varying humidity from 15% to 98% RH and recording the resistance at an interval of every 5 s. As seen in HR is the resistance at any given humidity, R0 is the starting resistance at 15% RH. The sensitivity factor comparison of the suspended and non-suspended sensors is shown in The humidity sensing mechanism of CNTs has been studied and discussed extensively in previous works . The senThe resistance of the suspended devices was measured at different temperatures to gauge the performance under different thermal operating conditions. In The long-term stability of a sensor is a major factor in determining its performance. To gauge long-term stability, the resistance of the suspended device was measured at regular intervals of seven to 10 days. The humidity was then flushed out and the sensor response was recorded. In this work, a humidity sensor based on a suspended carbon nanotube has been demonstrated. The suspended sensor showed good performance with near-zero hysteresis, fast response and recovery times, and exhibited good long-term stability. To demonstrate the advantages of using a suspended architecture, the performance of suspended CNTs was compared to a non-suspended sensor under similar testing conditions. Evidently, the suspended sensors showed superior humidity sensing characteristics such as near-zero hysteresis, and significantly improved sensitivity owing to lack of substrate effects and more surface area for adsorption. Moreover, the work improved the response time of the devices as compared to our previous work in  by more


HPV 18 is one of the most prevalent oncogenic types, only second to HPV 16, and included in the licensed vaccines on the market. In this study, we describe the production and characterization of a panel of monoclonal antibodies (mAb) to HPV18.The immunocompetence of 1B1 and 4C2 mAbs for HPV L1 protein was evaluated by SDS-PAGE analysis, neutralization assays, affinity identification, and ELISA. The 1B1 and 4C2 genes were sequenced and analyzed. Finally, the detection kit with the two mAbs was assessed for linearity, repeatability and specificity.Both mAbs specifically recognized HPV18 L1 and virus-like particles (VLPs). These mAbs are conformation-neutralizing antibodies that have high affinity and type specificity. Based on these characteristics of these mAbs, we developed an ELISA kit for specifically detecting HPV 18 antigen. We showed that this kit displayed good linearity, repeatability and sensitivity for detecting HPV18 L1 pentamer and HPV18 VLP.We characterized two monoclonal neutralizing antibodies for HPV L1 protein, and developed an ELISA kit for specifically detecting HPV 18 antigen. This newly developed kit can be used to monitor the potency of HPV vaccines throughout the entire production process as well as preliminary analysis of HPV18 infections. Human papillomaviruses (HPVs) are a group of non-enveloped viruses with a double-stranded DNA , 2, whicThere is an early-gene and a late-gene region within the genome of HPV virus. The early genes E1-E7 play roles in viral life cycle and pathogenesis, whereas the late genes L1 and L2 code for the viral capsid proteins L1 and L2 respectively –11. L1 i® (Merck) — a quadrivalent vaccine — targets two oncogenic types (HPV16 and HPV18) and two types that cause genital warts (HPV6 and HPV11)  for 30 min and boiling for 5 min. The 96-well microtiter plates were coated with L1-VLP (200 ng per well) overnight at 4 °C. After drying, the plates were blocked with 2% BSA (300 μl/well) for 2 h, and incubated with antibodies (100 μl at 0.3 μg/ml) for 1 h at room temperature. HRP-conjugated goat anti-mouse IgG (1:4000 in assay diluent) was added to the dried plates and incubated for 1 h at room temperature. The plates were washed with PBS 5 times prior to the addition of the chromogenic substrate solution (100 μl/well). The reaction was stopped by the addition of the termination solution (100 μl/well) after 10-min incubation in the dark, and the absorbance was measured at 450 nm.Neutralization assays with HPV18 pseudovirus (PsV18) were performed in 293FT cells as described previously .All mAbs were diluted to a nominal concentration of 200 μg/ml, and each mAb was further diluted by a 4-fold serial dilution. 50 μl PsV18 (at a pertinent concentration) was incubated with equal volume of mAbs for 1 h at 4 °C; PsV18 in PBS was used as the negative control. Subsequently, the antibody-pseudovirus mixture was incubated with 293FT cells . Seventy-two hours post seeding, the cells were harvested, and subjected to a flow cytometer to measure the fluorescence. The fluorescence inhibition rate was calculated as follows:50 and IC90) was determined for neutralization titer of the monoclonal antibody against PsV18.A 50 and 90% inhibited concentration (ICHPV18 L1-VLP antigen (10 ng/well) was incubated in 96-well plates overnight at 4 °C. The plates were washed and blocked with 2% BSA for 2 h at room temperature. Subsequently, 100 μl international standard  at 0.5 IU/ml concentration and equal volume of HRP-conjugated antibodies (0.3 μg/ml) were added to each well, with PBS as a negative control. Plates were incubated for 1.5 h at room temperature. The reaction was stopped, developed, and the absorbance was measured at 450 nm. The calculation of inhibition rate followed the equation below:Ka, the dissociation constant Kd and the binding affinity KD were calculated.In order to reflect the degree of binding of the antibody to the HPV18 L1-VLPs, the antigen-antibody binding and dissociation were analyzed using a BIACORE3000 biosensor ; the binding constants According to the manufacturer’s instructions, the HPV18 L1-VLPs was diluted to a concentration of 40 μg/ml , and coupled to the chip with the coupling level of 4000 RU.The antibodies were respectively diluted to seven concentrations in PBS, ranging from 0.3125 to 20 nM. After sampling for 60 s, binding for 60 s and dissociating for 500 s, the chip was regenerated using acetic acid/sodium acetate buffer, pH 5.0. According to the manufacturer’s instructions, the binding kinetics data were analyzed using Biacore 3000 Evaluation program.Two hybridoma cell lines (1B1 and 4C2) were harvested, and total mRNA was extracted, from which cDNA was synthesized by RT-PCR. The cDNA was amplified with high fidelity using variable region universal primers, and the PCR products were inserted into T carrier separately to determine their DNA sequences. The DNA sequences were translated into amino acid sequences that were aligned and analyzed subsequently.The 1B1/4C2 antibody pairing experiment using the sandwich ELISA method was performed to confirm the coating antibody and the detecting antibody respectively. The antigens were diluted to concentrations of 10μg/ml, 3μg/ml, lug/ml, 0.3μg/ml and 0.1μg/ml respectively, and the ELISA experiments were performed as described to identify batch-to-batch and inter-batch variations. The assays were repeated 10 times for each sample.The coating antibody 4C2 was diluted to a concentration of 10 μg/ml in 0.05 M sodium carbonate buffer, pH 9.6. 96-well plates were incubated overnight at 4 °C with 100 μl/well coating antibody. The plates were washed with PBST twice and blocked with 3% BSA (100 μl/well) at 37 °C. Two hours post seeding, the plates were washed with PBS, and protected with 10% aqueous sucrose solutions for 1 h at room temperature. The dried plates were sealed in vacuum aluminum foil bags and stored at 4 °C.The HRP-conjugated antibodies were named 1B1-HRP. 96-well plates were incubated for 1.5 h at 37 °C with 100 μl/well HPV18 L1-VLPs. After washing, 100 μl 1B1-HRP at 0.5 μg/ ml was added to each well, and allowed to react with the HPV18 L1-VLPs for 1 h at 37 °C. The dried plate was developed for 10 min at 37 °C prior to the addition of the termination solution (50 μl/well). The absorbance was measured at 450 nm.The specificity test of the Detection Kit was performed as follows: native L1-VLPs were denatured by incubation in denaturation buffer  for 30 min at room temperature and boiling for 5 min. Each well of a 96-well plate was used to test 3 μg HPV18 L1-VLPs in PBS.A total number of 10 mAbs against HPV18 L1-VLPs were developed from mouse hybridoma cells following the standard method, two of which were used to construct the HPV18 Detection Kit. To avoid any confusion, we only present data regarding to these two mAbs.Both of the purified mAbs 1B1 and 4C2 were analyzed by SDS-PAGE electrophoresis, where two bands were observed in each sample lane. The molecular weights of the two bands were 25 kDa and 50 kDa respectively, corresponding to the light- and heavy-chain of the antibody respectively. The purity of both mAbs was more than 95%  were measured by indirect ELISA. Both native and denatured L1-VLPs were used to determine whether they were conformational epitope-specific antibodies. Our results showed that both 1B1 and 4C2 mAbs reacted with native HPV18 L1-VLPs, and had a signal of > 1; whereas the OD0.1 Fig. . These r50 and IC90  by competitive ELISA assay. By applying the International Standard to the HPV18 L1-VLP coated plates prior to the application of our mAb, any epitopes recognized by both the International Standard and our mAb would be blocked by the International Standard, which would result in fewer mAb binding in the next step, thus a lower OD value than that of the control group. This experiment would reveal the correlation of the epitopes recognized by our mAbs with those by the International Standard – the higher the inhibition rate, the more similar the epitopes recognized by our mAb to those by the International Standard. As shown in Table Ka, disassociation constant Kd, and the binding affinity KD for each reaction , and calculated the binding constant To identify the mAbs obtained, mAbs 1B1 and 4C2 were sequenced. Amino acid sequence alignments of the light- and heavy-chains of these two mAbs are shown in Fig. Using 4C2 as the coating antibody and HRP-conjugated 1B1 as the detecting antibody, the linearity range of the Detection Kit was determined. The Curve was fitted with Four Parameter Equation, and as shown in Fig. The specificity of the Detection Kit was determined using both native and denatured L1-VLPs of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58. Amongst all the samples used, only native HPV18 L1-VLPs gave a positive signal Fig. . The resIn general, when developing and selecting mAbs for a detection kit of HPV18, we seek to satisfy four criteria. First, the sequence of each mAb must be a specific sequence in the kit. Second, the mAbs should have good binding affinity towards the antigen and would not rapidly dissociate . Third, In this study, two mAbs (1B1 and 4C2) were screened from 10 mAbs expressed by hybridoma cells stably expressing type-specific neutralizing antibodies against HPV18 L1-VLPs. A few features of mAbs 1B1 and 4C2, including antibody isotype, HPV type specificity, conformational epitopes, neutralization ability, and sequences were characterized. Both 1B1 and 4C2 had good specificity towards native HPV18 L1-VLPs and showed no cross-reactivity to the L1-VLPs of the other eight HPV types tested. Compared with antibodies described in similar studies , 15 that® 9 has the widest protection range. Our mAbs, which were demonstrated to have high titers only towards HPV18 L1-VLPs within the 9 HPV types analyzed , can be used in the specific detection of HPV18 VLPs in HPV vaccines with up to 9 valences or compounds that have HPV18 component. Furthermore, they can be applied to the detection of HPV18 infections in clinic.Amongst all the HPV vaccines currently available, the nonavalent GardasilThis pair of mAbs was subsequently used to develop an HPV18 Detection Kit. One mAb coated on the ELISA plate would capture the L1-VLPs, whereas the other mAb with conjugated HRP was used for detection. Such sandwich ELISA approach uses two mAbs to detect and quantify HPV18, allowing the detection of not only HPV18 L1 pentamers, but also HPV18 L1-VLPs. The detection limit of the kit was 0.l μg/ml, with a linear range of 0.l – 10 μg/ml. Assays have also confirmed the kit had high specificity and sensitivity only towards native HPV18 L1-VLPs. This Detection Kit has been applied in our routine quality control tests of HPV vaccine productions, and it was also used by other organizations in relevant analyses.This Detection Kit was designed primarily for the quality control throughout HPV vaccine production as well as preliminary detection of HPV types. We have taken this research further by developing a more sophisticated IHC system using these mAbs, aiming for a more accurate and convenient detection of HPV18 infections in tissue samples (to be reported elsewhere).In summary, high-affinity and strongly neutralizing antibodies were generated, characterized, and used to develop a sandwich ELISA kit that could identify and quantify HPV18 type-specific L1-VLP antigens within the nonavalent HPV vaccine range (HPV6/11/16/18/31/33/45/52/58). With the two mAbs we produced, the Detection Kit could detect the presence of HPV18 L1 pentamer or HPV18 L1-VLPs from up to nine different types of HPVs, thus could be widely used in quality control for clinical testing and vaccine manufacturers. This study has also provided us a guideline for the rational design of high-affinity monoclonal antibodies that could be applied to the diagnosis and treatment of HPV18 infections.