Mastercam Change Recognition

[Johanne]

Establishedin 1997, Grayco Machine Ltd., in Leduc, Alberta, Canada, manufactures parts for the agricultural and oil industries. Using Mastercam as a robust CAD/CAM platform for manufacturing efforts, the company specializes in producing pinions and gears for pump jacks as well as wireline winches for oil drilling rigs.

Despite ongoing volatility in the oil industry, the jobber shop is staying afloat thanks to the vision of its owner, Graham Peterson. A Red Seal journeyman machinist and longtime proponent of machining and apprenticeship programs, Peterson understands the need for skilled machinists. All Grayco employees are highly skilled Red Seal journeyman machinists well-versed in both manual and CNC machine operations.

To manufacture gears, programmers use a wire EDM coupled with CAM software from Mastercam. From basic 2-axis contouring to complex 4-axis motion, Mastercam Wire software supports powerful wirepaths and efficient cutting techniques. Full 3D CAD modeling, automatic lead-in and lead-out strategies, and streamlined multiple-part cutting are features of the flexible software package. Wire software permits control over wire motion, angle, entries, exits, and more. File tracking and change recognition allow for simple programming and reprogramming. Job elements can be modified and wirepaths updated without restarts.

Turnaround times are a driving force here. Incoming work requires one-up or two-up part production. A customer idea goes from pad and paper, through Mastercam and into the finished part within a short timeframe.

CAM software offers Grayco the flexibility to manufacture a variety of part geometries and sizes. If a piece is too large to fit on a machine, for example, an operator can work on one corner of the part at a time. He can program that corner, rotate the part, turn the model with Mastercam, re-post it, and go again.

Grayco contracts out surface modeling of its herringbone gears but plans to move production to milling machines. Current mill feeds are 200 hundred inches per minute; Grayco plans to purchase a machine that can accommodate 300 surface feet per minute with 20,000 RPM speeds to achieve required surface finish accuracy.

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Interleukins in body fluids usually circulate in tiny amounts (nano- and picomolar) and their concentrations can increase up to 1000-fold when immune system activation is required. Interleukin-6 (IL-6), a pleiotropic cytokine, is produced by various types of normal and cancer cells and is implicated in the stimulation of tumor cell proliferation, malignant transformation, and tumor progression6. Additionally, it has been reported that a high circulating IL-6 level might predict an inferior response to chemotherapy and therefore is an independent prognostic marker for breast cancer7 and lung cancer-specific survival, especially for those who receive chemotherapy8. Interleukin-8 (IL-8), an inflammatory cytokine, may also play an important role in breast cancer. So far, there have been only a few research papers concerning this cytokine. Yokoe et al. have measured serum IL-8 levels in 12 heavily pretreated patients with recurrent breast cancer and reported a small increase of IL-8 in those patients with refractory progressive disease and almost no decrease in those with partial response or no change after systemic therapy9. Interleukin-18 (IL-18) plays an important role in the T-cell-helper response. It acts as an angiogenic factor and a tumor suppressor. Some clinical studies have shown that the serum IL-18 level may be a prognostic factor in patients with gastric carcinoma, hematological malignancies, and metastatic breast cancer.

The particular interleukins IL-6, IL-8, and IL-18 are known to play a diverse role in breast cancer initiation and progression. However, only a few sensors for these immune markers have been proposed in the literature and until now there are no methods which allow the simultaneous quantification and multiplex analysis of interleukins in the body fluids. The development of such methods is extremely important. The complications caused by the presence of interfering compounds in a given sample form a major drawback in existing molecular sensor technologies, particularly in multi-analyte systems. SERS as a molecular fingerprinting technique has the ability to resolve analytes within mixtures. Current successes in the development of nanotechnologies and instrumentations have led to recognition of biomolecular systems based on SERS with higher sensitivity and chemical specificity10,11,12.

The SERS technology can be used potentially for the quantitative measurement of analytes with ultrahigh sensitivity and offers nondestructive, reliable, and fast detection of samples, which leads to various practical applications in studying, e.g. nucleic acids and proteins13, therapeutic agents14, drugs and trace materials15, and bacteria cells16, 17.

The multiplexed analysis (named parallel and simultaneous) based on a sandwich type SERS immunoassay is schematically illustrated in Fig. 1. The first layer of this sandwich structure is composed of immobilized antibodies against IL-6, IL-8, and IL-18 interleukins captured on a Ag-Au bimetallic SERS-active surface via 6-amino-1-hexanethiol (AHT) layers. This particular SERS substrate fulfills all the necessary requirements for biological, medical and analytical analysis, such as high sensitivity, stability, biocompatibility, and reproducibility18. The second layer comprises the complementary interleukins (proteins) captured by the selective antibodies. The third layer consists of Raman reporters (FC, p-MBA, and DTNB)-and antibodies-labeled immune-Au-nanoparticles. In the parallel approach (Fig. 1C), the individual SERS-active platform was functionalized using antibodies against selected interleukins: anti-IL6, anti-IL8, and anti-IL18, respectively. A microfluidic SERS-device with three chambers allows parallel detection and analysis of three selected interleukin levels in blood plasma. In the simultaneous multiplexed detection method (Fig. 1D), the SERS-active platform was functionalized with three antibodies, anti-IL6, anti-IL8, and anti-IL18 to permit the simultaneous detection of three target interleukins within the platform. A detailed description of the performance of the microfluidic SERS device is presented in Supplementary Materials, Chapter 5.

All experiments were performed in compliance with the relevant laws and institutional guidelines. The protocol of study was approved by the Ethics and Bioethics Committee of Cardinal Stefan Wyszyński University in Warsaw. Informed consent was obtained from all patients.

In our experiments we used human blood samples from 10 healthy volunteers available by courtesy of the Regional Blood Center. The samples underwent morphological analyses prior to use and revealed no abnormalities. Informed consent was obtained from all patients. All experiments were performed in compliance with the relevant laws and institutional guidelines. The protocol of study was approved by the Ethics and Bioethics Committee of Cardinal Stefan Wyszynski University in Warsaw.

The SERS spectra were prepared for principal component analysis (PCA) using a two-step approach. First, using an OPUS software (Bruker Optic GmbH 2012 version) the spectra were smoothed with a Savitsky-Golay filter, the background was removed using baseline correction, and then the spectra were normalized using a so-called Min-Max normalization. Then all the data were transferred to Unscrambler software (CAMO software AS, version 10.3, Norway) where the PCA analysis was performed. PCA is a multivariate technique that reduces the dimensionality of complex spectroscopic data from many wavenumber assignments to a few principal components (PCs) making it easier to identify the majority of variations within the spectra19.

In the present work PCA was carried out on three different data sets, consisting of spectra obtained for three types of studied immunocomplexes encoded by three types of Raman reporters (FC, p-MBA, and DTNB).

The selected interleukin antibodies anti-IL6, anti-IL8, and anti-IL-18 were immobilized onto the Ag-Au SERS-active substrate described above via 6-amino-1-hexanethiols layers using EDC/NHS chemistry (see Supplementary Materials). In the next step, the SERS activities of Raman reporters, i.e. FC, p-MBA, and DTNB-labeled immune Au nanoparticles were examined.

Immunoassays may come in many different formats and variations, but usually run in multiple steps with reagents being added and washed away or separated at different points in the assay30, 31. Such a strategy may cause problems which complicate the interpretation of data and may lead to wrong diagnoses. In the proposed SERS-based immunoassay we solve this difficulty via applying the designed microfluidic device. The signals could be collected from the same point on the SERS-assay (Fig. 1), which additionally enhances the reproducibility of the SERS-based immunoassay method. Moreover, the incorporation of SERS-active nanostructures into a microfluidic device offers a significantly larger active surface for immune reactions and hence improved performance of the immunoassay.

The excellent reproducibility is due to applying the microfluidic device that enables performing and monitoring the subsequent and controlled immunoreactions. Moreover, the proposed architecture allows for the collection of SERS signals from one spot on a SERS-active platform during the whole process of detection.

These data reveal that the newly developed SERS-based immunoassay offers a new recognition bioplatform for quantitative and simultaneous analysis of three interleukins: IL-6, IL-8, and IL-18 in body fluids with desired sensitivity and reproducibility.

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