Programmable nano-bio-chips: multifunctional clinical tools for use at the point-of-care

Abstract

A new generation of programmable diagnostic devices is needed to take advantage of information generated from the study of genomics, proteomics, metabolomics and glycomics. This report describes the 'programmable nano-bio-chip' with potential to bridge the significant scientific, technology and clinical gaps through the creation of a diagnostic platform to measure the molecules of life. This approach, with results at the point-of-care, possesses capabilities for measuring such diverse analyte classes as cells, proteins, DNA and small molecules in the same compact device. Applications such as disease diagnosis and prognosis for areas including cancer, heart disease and HIV are described. New diagnostic panels are inserted as 'plug and play' elements into the modular platform with universal assay operating systems and standard read out sequences. The nano-bio-chip ensemble exhibits excellent analytical performance and cost-effectiveness with extensive validation versus standard reference methods (R(2) = 0.95-0.99). This report describes the construction and use of two major classes of nano-bio-chip designs that serve as cellular and chemical processing units, and provides perspective on future growth in this newly emerging field of programmable nano-bio-chip sensor systems.

Figure 1. Bridging gaps in healthcare: from nanometer to global

The two CPU types explained in detail. The membrane-based, cellular processing unit (upper scheme (i)), is typified by cell counting applications, while the bead-based, chemical processing unit (lower scheme (ii)) has demonstrated utility in protein assays. Both utilize attributes of material across many different size scales. Nanometer-scale gaps between c.Ab. and d.Ab., and signaling probe (A & B) create bioconjugates for analyte fluorescence labeling (C) within the two CPU types (D). Expanded view in (B) illustrates the nanometer-scale network of capture agents. The modular labcard (E) houses the complete assay assembly for use in a portable, self-contained analyzer (F). Tracking of results of a global basis can become possible as these networked analyzers are distributed across the planet and secure diagnostic information that can be tied to geolocation. c.Ab: Capture antibody; d.Ab: Detecting antibody.

Figure 2. Compact, integrated analyzer

(A) Schematic of nano-bio-chip analyzer that is currently in development at LabNow. Assays completed with nano-bio-chip are performed with the compact, toaster-sized analyzer. The biochemical reactions are performed within the compact labcard (inset (A)). The standalone analzyer core (B) contains LED excitations source, actuators for fluid handling and a digital camera for image capture. The complete device and labcard (C) create an integrated, reaction approach.

Figure 3. Chemical processing unit

(A) The bead array is an image-based sensor that yields data in the form of a fluorescent photomicrograph. Various array sizes may be used including 4 × 5, 10 × 10 and the 3 × 4 shown here for a carcinoembryonic antigen assay (exposure time 1 s). Data collected correlates well (R² > 0.94) for both (B) saliva and (C) serum samples [35].

Figure 4. Cellular processing unit

Representative photomicrographs of whole blood labeled with a CD-specific antibody and quantum dot (QD) fluorescently tagged secondary antibody taken with a 10× objective and 3 s of exposure time. The QD 565 labels CD4⁺ cells including monocytes and T lymphocytes in the green channel (A) while QD 655 stains CD3⁺ lymphocytes red (B), as observed through separate filter cubes specific to each fluorophore. A digital overlap of the red and green images (C) shows monocytes (CD3⁺CD4⁺, green), T lymphocytes (CD3⁺CD4⁺, yellow) resulting from signal both in red and green channels, and remaining NK and CD8⁺CD3⁺ T-killer lymphocytes (CD3⁺CD4⁺, red). An alternative approach (D) utilizes a long-pass emission filter cube (520 nm and longer) allowing a single capture event to produce a similar image to that generated by separate photomicrographs. This membrane-based method was used to analyze a large sample set and found to correlate to flow cytometry for total lymphocyte counts at R² = 0.93 and T lymphocyte counts at R² = 0.97 (E) [36]. TLC: Total lymphocyte count.

Figure 5. Programming the nano-bio-chip

(A) Various hardware and software assay elements are programmed to result in the ideal combination suitable for analysis. The table in (A) highlights some of the experimental parameters that are optimized individually leading to ideal assay behavior in different nano-bio-chip programs. In addition to programming these parameters, the spatial code of beads placement and hence, molecular-level code of bioligands on the beads, is used to create programs specific to disease types. In (B) an assay for one analyte (blue) with control beads (red) is seen; (C) extends this to three different analytes (purple and orange) with the same negative control. (D) Larger arrays are also used and include positive control and calibrator beads. All scale bars are 300 μm. *Each oral swab contains thousands of epithelial cells suspended in buffer. Volumes injected into nano-bio-chip varied between 50 and 500 μl. ^‡Validation studies used 1 ml Bacillus globigii at 6.2 μg/l. ^§Sample volumes of 550 μl were used at a concentration of 3.2 μM 18-mer.

Figure 6. Moore’s Law-type growth

The figure of merit detailed above describes tests per person per analytical system. Note the logarithmic increase in the past decades.