Printing multistrain bacterial patterns with a piezoelectric inkjet printer

Many studies involving interacting microorganisms would benefit from simple devices able to deposit cells in precisely defined patterns. We describe an inexpensive bacterial piezoelectric inkjet printer (adapted from the design of the POSaM oligonucleotide microarrayer) that can be used to "print out" different strains of bacteria or chemicals in small droplets onto a flat surface at high resolution. The capabilities of this device are demonstrated by printing ordered arrays comprising two bacterial strains labeled with different fluorescent proteins. We also characterized several properties of this piezoelectric printer, such as the droplet volume (of the order of tens of pl), the distribution of number of cells in each droplet, and the dependence of droplet volume on printing frequency. We established the limits of the printing resolution, and determined that the printed viability of Escherichia coli exceeded 98.5%.     

Piezoelectric inkjet printing of a cross-hatch immunoassay on a disposable nylon membrane

The development of a cost-effective method for manufacturing immunoassays is a key step towards their commercial use. In this study, a piezoelectric inkjet printer and a nylon membrane were used to fabricate a disposable immunoassay. Using a piezoelectric inkjet printer, a cross-hatch pattern of goat anti-mouse antibody (GαM) and rabbit anti-horseradish peroxidase (RαHRP) antibody were deposited on the nylon membrane. These patterns were subsequently treated with a solution containing rabbit anti-goat antibody labeled with horseradish peroxidase (RαG-HRP). The effectiveness of the immobilization process was examined using tetramethylbenzidine (TMB), which oxidizes in the presence of HRP to form a visible precipitate. Optical evaluation of the TMB precipitate was used to assess the precision of the features in the inkjet-printed pattern as well as antibody functionality following inkjet printing. Uniform patterns that contained functional antibodies were fabricated using the piezoelectric inkjet printer. These results suggest that piezoelectric inkjet printing may be used to fabricate low-cost disposable immunoassays for biotechnology and healthcare applications.     

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.^1-10 Recently, thermal inkjet printing has also been used for gene transfection.^8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.^8,9,11The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.^{8, 12} Cell viability after printing has been shown to be similar to standard cell plating methods^1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. ^8,9,11A standard HP DeskJet 500 printer was modified to allow for cell printing.^{3, 5, 8} The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.^13-15     

用改良消减杂交结合选择性PCR法构建老年性白内障消减cDNA文库

为构建含较多大片段的高质量的老年性白内障消减cDNA文库 ,利用生物素标记、磁珠分离的改良消减杂交法获得差异cDNA .利用选择性PCR法扩增其中大片段差异cDNA ,将其与T 载体进行T A连接并转化入大肠杆菌 ,成功构建老年性白内障消减cDNA文库 .共获得 4 0 0 0余个克隆 ,随机挑取的 2 2个克隆中 ,≥ 10 0 0bp的片段有 7个 ,占 31 8% ,≥ 75 0bp有 15个 ,占 6 8 2 % .将≥ 75 0bp的 15个克隆进行反向点杂交 ,排除其中假阳性克隆 ,阳性克隆经测序并与GenBank比较 ,得到 6个已知基因、1个新基因 ,6个已知基因中 4个为全长基因 ,说明所得cDNA片段较大 ,文库质量较高 .改良消减杂交法结合选择性PCR法可以快速有效地获得大片段高质量的消减cDNA文库 ,为进一步筛选、鉴定老年性白内障致病相关基因奠定了基础     

Application of inkjet printing technique for biological material delivery and antimicrobial assays

A modified commercial inkjet printer was developed to deliver biological samples. The active Escherichia coli cells were directly printed at precisely targeted positions on agar-coated substrates via this technique to generate complex bacterial colony patterns. Viable cell arrays with a high density of 400 dots/cm² were obtained without the addition of any surfactants or other chemicals. Moreover, an applicable example of multiple-layer inkjet printing technique was adapted to deposit bacteria and antibiotics for antimicrobial potential assays. After fluorescent E. coli cells were printed, gradient concentrations of water-soluble antibiotics were ejected onto them to determine its minimum inhibitory concentration (MIC) to test the antimicrobial activities. This approach simplifies the experimental manipulation by replacing laborious manual loading processes with automatically controlled printing procedures, which makes it a versatile tool for high-throughput applications.     

Characterization, physical location and expression of the genes encoding calcium/calmodulin-dependent protein kinases in maize (Zea mays L.)

The maize genomic sequence and cDNA encoding a calcium/calmodulin-dependent protein kinase homolog were isolated and identified. The deduced peptide (MCK2) from this cDNA shared high amino acid identity (91.2%) with maize MCK1. These two genes were physically mapped onto chromosomes by fluorescence in situ hybridization using the first introns of the genes as gene-specific probes. While the MCK1 gene was assigned to a locus on the long arm of chromosome 9, the MCK2 gene was localized to a locus on the long arm of chromosome 1. Both of these genes were expressed in roots, leaves, stems and flowers, and the expression patterns of MCK were verified by RNA in situ hybridization. These results indicated that MCK expression is temporally and spatially regulated during maize growth and development.     

小麦抗病基因表达谱中的文库构建与筛选方法研究

以抗白粉病品系“百农 32 17×Mardler”BC5F4为材料 ,构建了白粉病菌诱导的普通cDNA文库和抑制消减杂交(SSH)cDNA文库。分别对两文库进行了一定规模的测序 ,获得普通cDNA文库不重复ESTs 387条和SSHcDNA文库ESTs 76 0条。将获得的ESTs与GenBank序列进行了BLASTn、BLASTx同源性分析。结果表明 :在普通文库中 ,一些参与光合作用与核糖体构成等的基因出现频率较高 ,而获得的抗病相关基因则较少。消减文库在构建方法、抗病相关基因的富集等方面具有明显的优越性 ,是目前抗病基因表达谱研究中的较好方法。利用高密度点阵膜杂交技术对两文库的筛选结果表明 ,该方法具有相对简便易操作、杂交膜可反复使用等优点 ;但也存在mRNA及同位素用量大等问题。经筛选 ,消减文库中有 5 4 1%的功能已知ESTs为抗病相关基因 ,被证明参与了小麦抗白粉病反应     

Improving piezoelectric cell printing accuracy and reliability through neutral buoyancy of suspensions

The sedimentation and aggregation of cells within inkjet printing systems has been hypothesized to negatively impact printer performance. The purpose of this study was to investigate this influence through the use of neutral buoyancy. Ficoll PM400 was used to create neutrally buoyant MCF‐7 breast cancer cell suspensions, which were ejected using a piezoelectric drop‐on‐demand inkjet printing system. It was found that using a neutrally buoyant suspension greatly increased the reproducibility of consistent cell counts, and eliminated nozzle clogging. Moreover, the use of Ficoll PM400 was shown to not affect cellular viability. This is the first demonstration of such scale and accuracy achieved using a piezoelectric inkjet printing system for cellular dispensing. Biotechnol. Bioeng. 2012; 109: 2932–2940. © 2012 Wiley Periodicals, Inc.     

An amperometric glucose biosensor prototype fabricated by thermal inkjet printing

The prototype of an amperometric glucose biosensor was realized by thermal inkjet printing using biological and electronic water-based inks, containing a glucose oxidase (GOD) from Aspergillus niger and the conducting polymer blend poly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid) (PEDOT/PSS), respectively. The biosensor was fabricated microdepositing PEDOT/PSS and GOD, in sequence, on ITO-glass, by a commercial inkjet printer, with the help of a commercial software. High density microdots matrices were so-realized, with a calculated resolution of about 221 x 221 dpi (dot per inch). By means of a rapid and easy assay it was demonstrated that no activity loss occurred upon the printing of GOD, despite of the use of a thermal printhead. The device was encapsulated in a semipermeable membrane of cellulose acetate, applied by dip-coating, in order to prevent dissolution of the enzyme and/or PEDOT/PSS in water. The preliminary response of the electrode was measured in an aqueous glucose solution in the presence of ferrocenemethanol (FeMeOH) as a mediator, and resulted linear up to 60 mM in glucose. The best sensitivity value achieved was 6.43 microAM(-1) cm(-2) (447 nAM(-1) U(-1) cm(-2)). The characteristics of the device, and the possible performance improvements have been analyzed and discussed. The reported findings indicate that inkjet printing could be a viable instrument for the easy construction of a working biosensor via direct digital design using biological and conductive polymer based inks. Such an approach may be seen as an example of "biopolytronics".     

Controlled patterning of peptide nanotubes and nanospheres using inkjet printing technology.

Peptide nanostructures are expected to serve as a major tool in future nanotechnological applications owing to their excellent self-assembly properties, biological and chemical flexibility and structural simplicity. Yet one of the limiting factors for the integration of peptide assemblies into functional electro-organic hybrid devices is the controlled patterning of their assemblies. Here we report the use of inkjet technology for the application of peptide nanostructures on nonbiological surfaces. The aromatic dipeptides nanotubes (ADNT) which readily self-assemble in solution were used as an 'ink' and patterned on transparency foil and ITO plastic surfaces using a commercial inkjet printer. While inkjet technology was used in the past for the patterning of carbon nanotubes, it was not used for the deposition of biomolecular nanostructures. Furthermore, during the development of the application we were able to produce two types of nanostructures, i.e. nanotubes and nanospheres by the self-assembly of the same aromatic dipeptide, tertbutoxycarbonyl-Phe-Phe-OH (Boc-Phe-Phe-OH), under different conditions. Both spherical and tubular structures could be efficiently patterned on surfaces into predesigned patterns. The applications of such technology are discussed.     

Genomic sequence analysis and organization of BmKalphaTx11 and BmKalphaTx15 from Buthus martensii Karsch: molecular evolution of alpha-toxin genes

Based on the reported cDNA sequences of BmKalphaTxs , the genes encoding toxin BmKalphaTx11 and BmKalphaTx15 were amplified by PCR from the Chinese scorpion Buthus martensii Karsch genomic DNA employing synthetic oligonucleotides. Sequences analysis of nucleotide showed that an intron about 500 bp length interrupts signal peptide coding regions of BmKalphaTx11 and BmKalphaTx15. Using cDNA sequence of BmKalphaTx11 as probe, southern hybridization of BmK genome total DNA was performed. The result indicates that BmKalphaTx11 is multicopy genes or belongs to multiple gene family with high homology genes. The similarity of BmKalpha-toxin gene sequences and southern hybridization revealed the evolution trace of BmKalpha-toxins: BmKalpha-toxin genes evolve from a common progenitor, and the genes diversity is associated with a process of locus duplication and gene divergence.     

Use of interspersed repetitive sequences-PCR products for cDNA selection

In order to increase the efficiency of cDNA selection approaches, we describe the use of interspersed repetitive sequences-PCR (IRS-PCR) products to isolate genes from large-insert genomic clones. IRS-PCR is conducted on total yeast DNA containing a YAC of interest so that there is no need to purify the starting genomic clone. This enables the production of large amounts of genomic substrate for cDNA selection and allows the use of unstable YAC clones. Moreover, the hybridization of the IRS-PCR product to the cDNA clones after selection introduces a positive selection step. We tested these PCR products from YACs for the presence of exons, using cDNAs originating from seven different genes. In each case, at least one exon was present in the IRS-PCR product. We have applied this strategy to four YAC clones originating from the human X Chromosome (Chr). All the selected cDNAs, strongly positive with the IRS-PCR product, did indeed originate from a gene in the region covered by the YAC. In all cases, the previously known genes contained in the genomic clones have been isolated. In addition, we have isolated human genes that have already been described but not assigned to any chromosomal region.     

A quality-controlled microarray method for gene expression profiling

Gene expression profiling on microarrays is widely used to measure the expression of large numbers of genes in a single experiment. Because of the high cost of this method, feasible numbers of replicates are limited, thus impairing the power of statistical analysis. As a step toward reducing technically induced variation, we developed a procedure of sample preparation and analysis that minimizes the number of sample manipulation steps, introduces quality control before array hybridization, and allows recovery of the prepared mRNA for independent validation of results. Sample preparation is based on mRNA separation using oligo(dT) magnetic beads, which are subsequently used for first-strand cDNA synthesis on the beads. cDNA covalently bound to the magnetic beads is used as template for second-strand cDNA synthesis, leaving the intact mRNA in solution for further analysis. The quality of the synthesized cDNA can be assessed by quantitative polymerase chain reaction using 3'- and 5'-specific primer pairs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase. Second-strand cDNA is chemically labeled with fluorescent dyes to avoid dye bias in enzymatic labeling reactions. After hybridization of two differently labeled samples to microarray slides, arrays are scanned and images analyzed automatically with high reproducibility. Quantile-normalized data from five biological replica display a coefficient of variation 45% for 90% of profiled genes, allowing detection of twofold changes with false positive and false negative rates of 10% each. We demonstrate successful application of the procedure for expression profiling in plant leaf tissue. However, the method could be easily adapted for samples from animal including human or from microbial origin.     

Linear amplification of catalyzed reporter deposition technology on nylon membrane microarray

The application of microarray analysis to gene expression from limited tissue samples has not been very successful because of the poor signal qualityfrom the genes expressed at low levels. Here we discussed the use of catalyzed reporter deposition (CARD) technology to amplify signals from limited RNA samples on nylon membrane cDNA microarray. When the input RNA level was greater than 10 microg, the genes expressed at high levels did not amplify in proportion to those expressed at low levels. Compared to conventional colorimetric detection, the CARD method requires less than 10% of the total RNA used for amplification of signal displayed onto a nylon membrane cDNA microarray. Total RNA (5-10 microg), as one can extract from a limited amount of specimen, was determined to produce a linear correlation between the colorimetric detection and CARD methods. Beyond this range, it can cause a nonlinear amplification of highly expressed and low-abundance genes. These results suggest that when amplification is needed for any applications using the CARD method, including DNA microarray experiments, precaution has to be taken in the amount of RNA used to avoid skew amplification and thus misleading conclusions.