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202 KiB

Open AccessArticle

Detection of Carcinoembryonic Antigens Using a Surface Plasmon Resonance Biosensor

by Fengyu Su, Chunye Xu, Minoru Taya, Kimie Murayama, Yasuro Shinohara and Shin-Ichiro Nishimura

Sensors 2008, 8(7), 4282-4295; https://doi.org/10.3390/s8074282 - 18 Jul 2008

Cited by 28 | Viewed by 12312

Abstract

Carcinoembryonic antigen (CEA) is an oncofoetal cell-surface glycoprotein that serves as an important tumor marker for colorectal and some other carcinomas. In this work, a CEA immunoassay using a surface plasmon resonance (SPR) biosensor has been developed. SPR could provide label-free, real-time detection [...] Read more.

Carcinoembryonic antigen (CEA) is an oncofoetal cell-surface glycoprotein that serves as an important tumor marker for colorectal and some other carcinomas. In this work, a CEA immunoassay using a surface plasmon resonance (SPR) biosensor has been developed. SPR could provide label-free, real-time detection with high sensitivity, though its ability to detect CEA in human serum was highly dependent on the analytical conditions employed. We investigated the influences of various analytical conditions including immobilization methods for anti-CEA antibody and composition of sensor surface on the selective and sensitive detection of CEA. The results show that anti-CEA antibody immobilized via Protein A or Protein G caused a large increase in the resonance signal upon injection of human serum due to the interactions with IgGs in serum, while direct covalent immobilization of anti-CEA antibody could substantially reduce it. An optimized protocol based on further kinetic analysis and the use of 2^nd and 3^rd antibodies for the sandwich assay allowed detecting spiked CEA in human serum as low as 25 ng/mL. Furthermore, a self-assembled monolayer of mixed ethylene-glycol terminated alkanethiols on gold was found to have a comparable ability in detecting CEA as CM5 with thick dextran matrix and C1 with short flat layer on gold. Full article

(This article belongs to the Special Issue Bioanalysis in Vivo/in Vitro)

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Immobilization of anti-CEA antibody onto sensor chip CM5; (a) via anti- mouse IgG polyclonal antibody, (b) via Protein A, (c) via Protein G, and (d) direct covalent binding. Full article ">
Sensorgrams of detecting (a) 100 ng/mL of CEA in HBS buffer and (b) 10-fold diluted human serum in HBS buffer on the three kinds of surfaces: i) via anti-mouse IgG polyclonal antibodies, ii) direct covalent immobilization, and iii) unmodified reference surface. Full article ">
Kinetic analysis of CEA antibody and antigen interactions, detection of CEA antigens with a series of concentrations (800 ng/mL to 12.5 ng/mL): color lines are experimental sensorgrams, and black lines are simulated sensorgrams by using BIAevaluation 4.1. Full article ">
Sensorgram of detecting 6.2 ng/mL of CEA in HBS buffer. Full article ">
Sandwich assay of 20 ng/mL (a) and 1.0 ng/mL (b) of CEA in HBS buffer, and a series of CEA-spiked serum sample (c). Full article ">
Illustration of the sensor chip CM5, C1 and EG-SAM (a) and the detection of a series of CEA on C1 and EG-SAM (b). Full article ">
Signal shift as a function of concentration for three kinds of sensor chips; ◊ CM5, □ C1, and Δ SAM. Full article ">

170 KiB

Open AccessArticle

An Investigation on Micro-Raman Spectra and Wavelet Data Analysis for Pemphigus Vulgaris Follow-up Monitoring.

by Carlo Camerlingo, Flora Zenone, Giuseppe Perna, Vito Capozzi, Nicola Cirillo, Giovanni Maria Gaeta and Maria Lepore

Sensors 2008, 8(6), 3656-3664; https://doi.org/10.3390/s8063656 - 1 Jun 2008

Cited by 25 | Viewed by 13943

Abstract

A wavelet multi-component decomposition algorithm has been used for data analysis of micro-Raman spectra of blood serum samples from patients affected by pemphigus vulgaris at different stages. Pemphigus is a chronic, autoimmune, blistering disease of the skin and mucous membranes with a potentially [...] Read more.

A wavelet multi-component decomposition algorithm has been used for data analysis of micro-Raman spectra of blood serum samples from patients affected by pemphigus vulgaris at different stages. Pemphigus is a chronic, autoimmune, blistering disease of the skin and mucous membranes with a potentially fatal outcome. Spectra were measured by means of a Raman confocal microspectrometer apparatus using the 632.8 nm line of a He-Ne laser source. A discrete wavelet transform decomposition method has been applied to the recorded Raman spectra in order to overcome problems related to low-level signals and the presence of noise and background components due to light scattering and fluorescence. This numerical data treatment can automatically extract quantitative information from the Raman spectra and makes more reliable the data comparison. Even if an exhaustive investigation has not been done in this work, the feasibility of the follow-up monitoring of pemphigus vulgaris pathology has been clearly proved with useful implications for the clinical applications. Full article

(This article belongs to the Special Issue Bioanalysis in Vivo/in Vitro)

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Outline of Raman signal elaboration by wavelet algorithm. The raw data spectra (a and b) are decomposed by Discrete Wavelet Transform (DWT) in the components D1,…,D8, A8 (c and d). Using the Inverted Discrete Wavelet Transform (IDWT) the signal is reconstructed from D5,.., D8 components (e and f). Full article ">
Typical Raman spectrum of blood serum from patients with active PV (a), under drug therapy (b) and from a recovered patient (c). Full article ">
Behavior of R2 resulting from linear regression of Raman spectra relative to blood serum from patients in the remission stage of illness (recovered), from patients under drug therapy and from PV active patients in the wavenumber 1000-1800 cm-1. Dots and bar indicate the mean of R2 and the error in its determination. Full article ">
Typical Raman spectrum from under drug therapy (a) and PV active patients (b). The spectra are compared to the spectrum of recovered patient (thin lines) by means of linear regression analysis. The residual signal between signal in (a), signal in (b) and reference signal (from recovered patient) is reported in (c) and (d) respectively. Full article ">

230 KiB

Open AccessArticle

DNA Extraction Systematics for Spectroscopic Studies

by Bianca Fogazza Palma, Amanda Borges Ferrari, Renata Andrade Bitar, Maria Angélica Gargione Cardoso, Airton A. Martin and Herculano Da Silva Martinho

Sensors 2008, 8(6), 3624-3632; https://doi.org/10.3390/s8063624 - 1 Jun 2008

Cited by 4 | Viewed by 13276

Abstract

Study of genetic material allows the comprehension the origin of the many biochemical changes that follow diseases, like cancer, promoting the development of early preventive inquiry and more efficient individual treatments. Raman spectroscopy can be an important tool in DNA study, since it [...] Read more.

Study of genetic material allows the comprehension the origin of the many biochemical changes that follow diseases, like cancer, promoting the development of early preventive inquiry and more efficient individual treatments. Raman spectroscopy can be an important tool in DNA study, since it allows probe molecular vibrations of genetic material in a fast way. The present work established a systematic way for extract DNA in suitable concentrations and structural integrity allowing studies by Raman spectroscopy or other spectroscopic technique, including bio-analytical sensors for probing genetic alterations. Full article

(This article belongs to the Special Issue Bioanalysis in Vivo/in Vitro)

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UV absorption curves of extracted DNA with DNeasy, ChargeSwitch and Phe-Cl methods with 3-4 hours of digestion. Full article ">
Comparison between UV absorption spectra of different elution buffers. Full article ">
Agar gel electrophoresis 1% with ethidium bromide of extracted DNA from female breast cancer tissue. S1: 2 μL of 1Kb DNA Ladder standard sample (in base pairs-bp: 12,216; 11,198; 10,180; 9162; 8144; 7126; 6108; 5090; 4072; 3054; 2036; 1636; 1018; 506; 396; 344 and 298) A: 5 μL of DNA sample extracted by Qiagen DNeasy method; B: 5 μL of DNA sample extracted by ChargeSwitch method; C: 5 μL of DNA sample extracted by Phe-Cl method and S2: 2 μL of Hind III Fragments Standard sample (in base pairs: 23,130; 9416; 6557; 4361; 2322; 2027; 564 and 125). Full article ">
Comparison between Raman spectra from capillary tube and optical long path cell using Quartz and CaF2 windows. Full article ">
Comparison between Raman spectra of elution buffers. Full article ">
Comparison between Raman spectra from DNA extracted by DNeasy Qiagen (de-ionized water elution) method measured in optical long path cell and in capillary tube. Full article ">
Raman spectra of extracted DNA with Phe-Cl (black line), DNeasy (red line), and ChargeSwitch (blue line) methods measured in optical long path cell. Each curve is an average of four Raman spectra. The asterisks indicate bands from the optical cell. Full article ">
Raman spectra of DNA extracted with DNease method measured in optical long path cell. a) Data taken with quartz window. b) Data taken with CaF2 window. c) Average spectra after deconvolution. Full article ">

870 KiB

Open AccessArticle

Improvement of Aptamer Affinity by Dimerization

by Hijiri Hasegawa, Ken-ichi Taira, Koji Sode and Kazunori Ikebukuro

Sensors 2008, 8(2), 1090-1098; https://doi.org/10.3390/s8021090 - 19 Feb 2008

Cited by 143 | Viewed by 15575

Abstract

To increase the affinities of aptamers for their targets, we designed an aptamerdimer for thrombin and VEGF. This design is based on the avidity of the antibody, whichenables the aptamer to connect easily since it is a single-strand nucleic acid. In this study,we [...] Read more.

To increase the affinities of aptamers for their targets, we designed an aptamerdimer for thrombin and VEGF. This design is based on the avidity of the antibody, whichenables the aptamer to connect easily since it is a single-strand nucleic acid. In this study,we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer.Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimerhas a K_d value which is 1/10 of that of the monomers from which it is composed. Also, thedesigned aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers againstvascular endothelial growth factor (VEGF₁₆₅), which is a homodimeric protein. As in thecase of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower K_d value than that of the monomer. This study demonstrated that the dimerization of aptamerseffectively improves the affinities of those aptamers for their targets. Full article

(This article belongs to the Special Issue Bioanalysis in Vivo/in Vitro)

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Review

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422 KiB

Open AccessReview

Visible Genotype Sensor Array

by Yuichi Michikawa, Tomo Suga, Yoshimi Ohtsuka, Izumi Matsumoto, Atsuko Ishikawa, Kenichi Ishikawa, Mayumi Iwakawa and Takashi Imai

Sensors 2008, 8(4), 2722-2735; https://doi.org/10.3390/s8042722 - 17 Apr 2008

Cited by 12 | Viewed by 12235

Abstract

A visible sensor array system for simultaneous multiple SNP genotyping has been developed using a new plastic base with specific surface chemistry. Discrimination of SNP alleles is carried out by an allele-specific extension reaction using immobilized oligonucleotide primers. The 3’-ends of oligonucleotide primers [...] Read more.

A visible sensor array system for simultaneous multiple SNP genotyping has been developed using a new plastic base with specific surface chemistry. Discrimination of SNP alleles is carried out by an allele-specific extension reaction using immobilized oligonucleotide primers. The 3’-ends of oligonucleotide primers are modified with a locked nucleic acid to enhance their efficiency in allelic discrimination. Biotin-dUTPs included in the reaction mixture are selectively incorporated into extending primer sequences and are utilized as tags for alkaline phosphatase-mediated precipitation of colored chemical substrates onto the surface of the plastic base. The visible precipitates allow immediate inspection of typing results by the naked eye and easy recording by a digital camera equipped on a commercial mobile phone. Up to four individuals can be analyzed on a single sensor array and multiple sensor arrays can be handled in a single operation. All of the reactions can be performed within one hour using conventional laboratory instruments. This visible genotype sensor array is suitable for “focused genomics” that follows “comprehensive genomics”. Full article

(This article belongs to the Special Issue Bioanalysis in Vivo/in Vitro)

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Recording the visible genotype sensor array image using a digital camera equipped on a mobile phone. A: Image recording using a mobile phone. B: Recorded image on the mobile phone. Individual spots on the sensor array indicate target SNP allele types. Visibility of the genotyping spots allows immediate inspection of results. Full article ">
Structure of an allele-discriminating immobilized oligonucleotide. The 3′-end nucleotide opposing the target SNP nucleotide in the template DNA is LNA modified to enhance allelic discrimination efficiency. The Tm of the backbone oligonucleotide is set to be 60°C. Amino C6 is attached to the 5′-end nucleotide for covalent immobilization to the surface of a plastic base. Full article ">
Spotting of oligonucleotides on plastic bases. A: MassARRAY Nanodispensor. B: Spotting on a plastic base. C: Heating of the spotted plastic base. D: Comparison of original and spotted plastic bases. Full article ">
Post spotting processes in preparation of visible genotype sensor array. A: Attaching a Multiwell Geneframe onto each plastic base. B: Simultaneous handling of multiple plastic bases. C: Masking unspotted surface of plastic bases by soaking in 1 x S-BIO blocking solution. D: Removal of surface solution by centrifugation. Full article ">
Scheme of reaction processes on the visible genotype sensor array. Only three reaction processes are necessary. These reactions can be performed by conventional laboratory instruments shown in <a href="#f9-sensors-08-02722" class="html-fig">Figure 9</a>. Typing results can be visibly inspected within one hour. Full article ">
Allele-specific primer extension reaction. The immobilized oligonucleotide (Allele 1) that is hybridized to perfectly matched template (Allele 1 template) can be extended according to the sequence of the template. Biotin-dUTPs in the solution are thus incorporated during the extension process. Extension of the immobilized oligonucleotide (Allele 1) hybridized to a mismatched template (Allele 2 template) is efficiently inhibited by the LNA modification at its 3′ end. Full article ">
Binding of alkaline phosphatase-conjugated streptavidin to the incorporated biotin-dUTPs. Streptavidin specifically binds to biotin molecules in the extended sequence of the immobilized oligonucleotide. This reaction allows spatially restricted localization of the alkaline phosphatase-streptavidin conjugates on the plastic base. Full article ">
Colored substrate precipitation. Captured alkaline phosphatases catalyze conversion of soluble NBT into unsoluble, colored NBT-formazon. The resulting NBT-formazon precipitate adheres to the surface of the plastic base, giving visible spots. Full article ">
Overview of experimental set-up for reactions on the sensor array. A: Set-up of allele-specific primer extension. The reaction mixture was added to each well of the visible genotype sensor arrays then placed on a plastic tip case containing pre-warmed water. B: Allele-specific primer extension was performed in a constant temperature incubator at 65°C. The plastic tip case was wrapped with Saran wrap to preserve humidity. C: Incubation of alkaline phosphatase-conjugated streptavidin. D: Color development using BCIP/NBT. Full article ">

4388 KiB

Open AccessReview

Imaging In Mice With Fluorescent Proteins: From Macro To Subcellular

by Robert M. Hoffman

Sensors 2008, 8(2), 1157-1173; https://doi.org/10.3390/s8021157 - 22 Feb 2008

Cited by 19 | Viewed by 15204

Abstract

Whole-body imaging with fluorescent proteins has been shown to be a powerfultechnology with many applications in small animals. Brighter, red-shifted proteins can makewhole-body imaging even more sensitive due to reduced absorption by tissues and less scatter.For example, a new protein called Katushka has [...] Read more.

Whole-body imaging with fluorescent proteins has been shown to be a powerfultechnology with many applications in small animals. Brighter, red-shifted proteins can makewhole-body imaging even more sensitive due to reduced absorption by tissues and less scatter.For example, a new protein called Katushka has been isolated that is the brightest known proteinwith emission at wavelengths longer than 620 nm. This new protein offers potential for non-invasive whole-body macro imaging such as of tumor growth. For subcellular imaging, toobserve cytoplasmic and nuclear dynamics in the living mouse, cancer cells were labeled in thenucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Thenuclear and cytoplasmic behavior of cancer cells in real time in blood vessels was imaged as theytrafficked by various means or adhered to the vessel surface in the abdominal skin flap. Duringextravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cellsexited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasmand nuclei underwent deformation during extravasation. Cancer cells trafficking in lymphaticvessels was also imaged. To noninvasively image cancer cell/stromal cell interaction in the tumormicroenvironment as well as drug response at the cellular level in live animals in real time, wedeveloped a new imageable three-color animal model. The model consists of GFP-expressingmice transplanted with the dual-color cancer cells. With the dual-color cancer cells and a highlysensitive small animal imaging system, subcellular dynamics can now be observed in live mice inreal time. Fluorescent proteins thus enable both macro and micro imaging technology and thereby provide the basis for the new field of in vivo cell biology. Full article

(This article belongs to the Special Issue Bioanalysis in Vivo/in Vitro)

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1148 KiB

Open AccessReview

Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

by Nobuaki Soh

Sensors 2008, 8(2), 1004-1024; https://doi.org/10.3390/s8021004 - 19 Feb 2008

Cited by 75 | Viewed by 14171

Abstract

Site-specific chemical labeling utilizing small fluorescent molecules is apowerful and attractive technique for in vivo and in vitro analysis of cellular proteins,which can circumvent some problems in genetic encoding labeling by large fluorescentproteins. In particular, affinity labeling based on metal-chelation, advantageous due to [...] Read more.

Site-specific chemical labeling utilizing small fluorescent molecules is apowerful and attractive technique for in vivo and in vitro analysis of cellular proteins,which can circumvent some problems in genetic encoding labeling by large fluorescentproteins. In particular, affinity labeling based on metal-chelation, advantageous due to thehigh selectivity/simplicity and the small tag-size, is promising, as well as enzymaticcovalent labeling, thereby a variety of novel methods have been studied in recent years.This review describes the advances in chemical labeling of proteins, especially highlightingthe metal-chelation methodology. Full article

(This article belongs to the Special Issue Bioanalysis in Vivo/in Vitro)

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